Methods and compositions for topoisomerase i modulated tumor suppression

ABSTRACT

Disclosed herein are methods and compositions for determining the sensitivity or enhancing the sensitivity of cells to the effects of topoisomerase I inhibitors. Also disclosed are methods and compositions for inducing cell death, apoptosis and/or growth arrest which may be used for tumor suppression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of application Ser. No.12/377,498, filed Feb. 13, 2009, which is a 35 USC 371 National PhaseEntry Application from PCT/US07/018,387, filed Aug. 16, 2007, anddesignating the U.S., which claims the priority of U.S. ProvisionalApplication No. 60/822,774 filed Aug. 18, 2006, the disclosures of whichare incorporated herein by reference in their entireties.

RIGHTS IN THE INVENTION

This invention was supported in part by grants from the NIH/NCI (CA111868, CA 135369) and from the California Tobacco-Related DiseaseResearch Program (11RT-0074). The Government may have certain rights inthis invention.

FIELD OF INVENTION

This invention relates to the field of cancer therapy and diagnostics.

BACKGROUND OF INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Topoisomerase I is a nuclear enzyme that plays an important role in cellproliferation. The enzyme catalyzes the uncoiling of DNA duringreplication and transcription (Pommier, et al., Biochim Biophys Acta1998; 1400(1-3):83-105; Wang, Annu Rev Biochem 1996; 65:635-92).

The activity of topoisomerase I is regulated by phosphorylation. Suchphosphorylation occurs primarily on serine residues (Turman, et al.,Biochem Med Metab Biol 1993; 50(2):210-25; Coderoni, et al., Int JBiochem 1990; 22(7):737-46; Kaiserman, et al., Biochemistry 1988;27(9):3216-22; Samuels, et al., J Biol Chem 1992; 267(16):11156-62) andappears to be necessary for the initial complex formation between theenzyme and the DNA (Coderoni, et al., Int J Biochem 1990; 22(7):737-46).

Human cancers are characterized by uncontrolled proliferation ofabnormal cells. Topoisomerase I inhibitors have been used aschemotherapeutic agents that interfere with normal DNA replication andcell division. However, some cancers are not sensitive to suchtopoisomerase I inhibitors.

SUMMARY OF THE INVENTION

The present invention provides methods (and related compositions) forincreasing the sensitivity of cells (e.g., cancer cells) to the activityof topoisomerase I inhibitors. The invention also provides methods forinducing growth arrest and/or cell death in cells (e.g., cancer cells).Further, the invention provides methods for determining the sensitivityof a cell (e.g., a cancer cell) to the effects of a topoisomerase Iinhibitor.

The invention is based upon the discovery that cells resistant totopoisomerase inhibitors frequently have a deficiency in topoisomerase Iserine phosphorylation, rendering them less sensitive (or insensitive)to the cytotoxic effect of topoisomerase I inhibitors. The deficiency intopoisomerase I phosphorylation reduces the ability of topoisomerase Ito bind p14ARF (ARF), an activator protein. Thus, cancer cells can beassessed for their sensitivity to topoisomerase I inhibitors, prior toinitiating therapy, by measuring the level of serine phosphorylation oftopoisomerase I, its activity, and/or its ability to bind ARF. Likewise,cells can be sensitized to the effects of topoisomerase I inhibitors byincreasing the amount of serine phosphorylation of topoisomerase I, orby increasing ARF-topoisomerase I complex formation by increasing, forexample, the amount of ARF available for complexation with serinephosphorylated topoisomerase I.

An additional feature of the invention is the discovery that cell deathand/or growth arrest may be induced by disrupting ARF-topoisomerase Icomplex formation. It is believed that free ARF, released from theARF-topoisomerase I complexes, increases the biological activity of p53(a known tumor suppressor gene) by sequestering HDM2, a p53 inhibitor.

Accordingly, in one aspect, the invention provides a method forincreasing the sensitivity of a cell to a topoisomerase I inhibitor bycontacting the cell with an agent that increases the level oftopoisomerase I serine phosphorylation.

In another aspect, the invention provides a method for inducing cellkilling, apoptosis, and/or growth arrest in a cell by contacting thecell with an agent that increases the level of topoisomerase I serinephosphorylation, and further contacting the cell with a topoisomerase Iinhibitor.

In one embodiment, the agent increases the serine kinase biologicalactivity in the cell. Preferably, the serine kinase biological activityis increased in the nucleus of the cell, the nucleolus, or in theperi-nucleolar region. Suitable agents include, for example, serinekinase agonists, activators, and cofactors. Other agents include vectorsencoding a serine kinase enzyme, operably linked to a promoter.Preferably, the serine kinase phosphorylates topoisomerase I on at leastone serine residue (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more serineresidues), and wherein such phosphorylation is capable of promotingARF-topoisomerase I complex formation. Suitable serine kinases include,for example, casein kinase II also known as protein kinase CK2 (referredto throughout this application as CKII or CK2 interchangeably), orprotein kinase C (PKC).

In other embodiments, the cell is a cancer cell including, for example,a lung cancer cell, a prostate cancer cell, a hepatocellular carcinomacell, a breast cancer cell, a colorectal cancer cell, an acutemyelogenous leukemia cell, a melanoma cell, an ovarian cancer cell, aneuroendocrine carcinoma cell, a gastric cancer cell, an esophagealcancer cell, a pancreatic cancer cell, an adenocarcinoma cell, a braincancer cell, a head and neck cancer cell, a bone marrow-derived cancercell, a bone cancer cell, a kidney cancer cell, a retina cancer cell, abladder cancer cell, a liver cancer cell, and a mesothelioma cancercell. Preferably, the cell is present within a human patient.

In other embodiments, the cell is further contacted with at least oneother chemotherapeutic agent. Suitable chemotherapeutic agents include,for example, alkylating agents, anti-metabolites, vinca alkaloikds, andanti-tumor antibodies.

In other embodiments, the topoisomerase I inhibitor stabilizes atopoisomerase I-DNA complex. Preferable topoisomerase I inhibitorsinclude, for example, camptothecin, irinotecan, topotecan, and analogsthereof, for example, 9-aminocamptothecin, 9-nitrocamptothecin(Rubitecan, Oratecan, Belotecan), 10-hydroxycamptothecin, Lurtotecan,10,11 methylenedioxycamptothecin, Morpholinocamptothecin, Extatecan,Silatecan, Diflomotecan, Homocamptotehcin, BN80927, 20-hydroxy-linkedmodifications to camptothecin, and others discussed in Venditto andSimanek, Mol Pharmaceutics 2010; 7(2):307-349, as well asnon-camptothecin-derived topoisomerase I inhibitors that act similarlyto stabilize the topoisomerase I-DNA complex as discussed in Pommier,Chemical Reviews 2009; 109:2894-2902.

In another aspect, the invention provides, for a cell expressingincreased phosphorylation of topoisomerase I, a method for increasingthe sensitivity of a said cell to a topoisomerase I inhibitor bycontacting the cell with an agent that increases the ARF-topoisomerase Icomplex formation.

In another aspect, the invention provides, for a cell expressingincreased phosphorylation of topoisomerase I, a method for inducing cellkilling, apoptosis, and/or growth arrest in said cell by contacting thecell with an agent that increases ARF-topoisomerase I complex formation,and further contacting the cell with a topoisomerase I inhibitor.

In a related aspect, the invention provides a method for treating cancerin a patient (e.g., a human patient), who has been diagnosed as havingcancer, by administering to the patient an agent that increasesARF-topoisomerase I complex formation, and further administering to thepatient a topoisomerase I inhibitor

In one embodiment, the agent is a vector encoding ARF, or a biologicallyactive fragment thereof, operably linked to a promoter. Preferably, thebiologically active ARF fragment contains amino acid residues 66-84 ofARF.

In another embodiment, the agent increases the amount of topoisomerase Iserine phosphorylation.

In another embodiment, the agent increases the serine kinase biologicalactivity in the cell. Preferably, the serine kinase biological activityis increased in the nucleus of the cell, the nucleolus, or in theperi-nucleolar region. Suitable agents include, for example, serinekinase agonists, activators, and cofactors. Other agents include vectorsencoding a serine kinase enzyme, operably linked to a promoter.Preferably, the serine kinase phosphorylates topoisomerase I on at leastone serine residue (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more serineresidues), and wherein such phosphorylation is capable of promotingARF-topoisomerase I complex formation. Suitable serine kinases include,for example, casein kinase II (CKII) or protein kinase C (PKC).

In other embodiments, the agent is a vector encoding ARF, or abiologically active fragment thereof, operably linked to a promoter.Preferably, the biologically active ARF fragment contains amino acidresidues 66-84 of ARF.

In other embodiments, the cancer is, for example, lung cancer, prostatecancer, hepatocellular carcinoma, breast cancer, colorectal cancer,acute myelogenous leukemia, melanoma, ovarian cancer, neuroendocrinecarcinoma, gastric cancer, esophageal cancer, pancreatic cancer,adenocarcinoma, adenocarcinoma, brain cancer, head and neck cancer, bonemarrow-derived cancer, bone cancer, kidney cancer, retina cancer,bladder cancer, liver cancer, or mesothelioma cancer.

In other embodiments, the patient is further administered with at leastone other chemotherapeutic agent including, for example, an alkylatingagent, an anti-metabolite, a vinca alkaloikd, or an anti-tumor antibody.In other embodiments, the patient is administered anti-cancer radiationtherapy prior to, concurrent with, or subsequent to administration ofthe topoisomerase I inhibitor.

Suitable topoisomerase I inhibitors stabilize the topoisomerase I-DNAcomplex. Preferable topoisomerase I inhibitors include, for example,camptothecin, irinotecan, topotecan, and analogs thereof, as well asnon-camptothecin-derived topoisomerase I inhibitors that act similarlyto stabilize the topoisomerase I-DNA complex.

As used herein, “topoisomerase I” refers to human topoisomerase I foundat Gen bank accession no NM_(—)003286 (FIG. 10).

As used herein, “p14ARF (ARF)” refers to the human ARF protein found atGenbank accession no. NP_(—)478102 (FIG. 9) and its homologs. It isbelieved that ARF interacts with, and activates, topoisomerase I.

Biologically active fragments of ARF contain substantially all of thetopoisomerase binding domain (i.e., amino acid residues 66-84)responsible for topoisomerase I binding. In all cases, the ARFpolypeptide must be capable of binding to phosphorylated topoisomeraseI. Suitable biologically active fragments include, for example, anN-terminal truncation of the ARF protein (e.g., amino acid residues66-132), or a polypeptide fragment or chimeric protein containingsubstantially all of the topoisomerase I binding domain (amino acidresidues 66-84).

By “serine kinase biological activity” is meant any enzymatic activitythat is capable of phosphorylating a serine amino acid residue on atarget protein. Typically, this is an ATP-dependent reaction in whichthe γ-phosphate group of an ATP molecule is transferred to the serineresidue of the substrate protein. Preferred serine kinases include, forexample, CKII and PKC.

By “increased serine kinase biological activity,” when referring to theserine kinase biological activity within a cell in accordance with theprinciples of this disclosure, is meant a level of serine kinasebiological activity in the cell nucleus which, following a specifictreatment or intervention, is higher than would otherwise be present inthe same cell absent that specific treatment or intervention (i.e., thebasal level). Elevated serine kinase biological activity is preferablyat least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, or more greater thanthe basal serine kinase biological activity level. Elevated serinekinase biological activity is determined using an assay which directlymeasures phosphorylation events attributable to the kinase activity.

A suitable assay for Protein Kinase C (PKC) can be carried out using aPKC assay kit from Upstate Biotechnology/Millipore (Temecula, Calif.).Cell lysates are prepared by lysing cells in extraction buffer (50 mMHEPES [pH 7.5]. 150 mM NaCl, 0.1% Tween 20, 1 mM EDTA, 2.5 mM EGTA, 10%glycerol) that contains protease inhibitors (10 μg of aprotinin per ml,10 μg of leupeptin per ml, 0.1 mM phenylmethylsulfonyl fluoride) andphosphatase inhibitors (1 mM NaF, 0.1 mM Na₃VO₄, 10 mM1′-glycerophosphate) as described in Soh, et al., Molecular and CellularBiology 1999; 19:1313-1324. 10 μg of cell lysate is then assayed in thepresence of assay buffer supplied in the kit, specific PKC substratepeptide [QKRPSQRSKYL], and γ-[³²P]-ATP (Perkin Elmer, Waltham, Mass.)for 15 minutes at 30° C., as per the instruction manual. The finalreaction conditions are as follows: 3.3 mM MOPS pH 7.2, 4.2 mMβ-glycerol phosphate, 0.17 mM sodium orthovanadate, 0.17 mMdithiothreitol, 0.17 mM CaCl₂, 83 μM specific substrate peptide, 0.33 μMPKA inhibitor peptide, 3.3 μM compound R24571, 80 μg/mlphosphatidylserine, 8 μg/ml, 83 μM ATP, 10 μCi γ-[³²P]-ATP. The phosphorylatedsubstrate is then separated from residual γ-[³²P]-ATP using supplied P81phosphocellulose paper and quantitated by scintillation counter.

A suitable assay for CKII can be carried out using a CKII assay kit fromUpstate Biotechnology/Millipore (Temecula, Calif.). Cell lysates areprepared by lysing cells in extraction buffer (50 mM HEPES [pH 7.5], 150mM NaCl, 0.1% Tween 20, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol) thatcontains protease inhibitors (10 μg of aprotinin per ml, 10 mg ofleupeptin per ml, 0.1 mM phenylmethylsulfonyl fluoride) and phosphataseinhibitors (1 mM NaF, 0.1 mM Na₃VO₄, 10 mM β-glycerophosphate) asdescribed in Soh, et al., Molecular and Cellular Biology 1999;19:1313-1324. 10 μg of cell lysate is then assayed in the presence ofassay buffer supplied in the kit, specific CKII substrate peptide[RRRDDDSDDD], and γ-[³²P]-ATP (Perkin Elmer, Waltham, Mass.) for 15minutes at 30° C., as per the instruction manual. The final reactionconditions are as follows: 4 mM MOPS pH 7.2, 5 mM β-glycerol phosphate,1 mM EGTA, 0.2 mM Na orthovanadate, 0.2 mM dithiothreitol, 200 μMspecific substrate peptide, 0.4 mM PKA inhibitor peptide, 90 μM ATP, 10μCi γ-[³²P]-ATP. The phosphorylated substrate is then precipitated byadding trichloracetic acid (TCA) to 10%, and quantitated byscintillation counting.

The skilled artisan recognizes that there exist a variety of kinaseassays for measuring the activity of PKC, CKII, as well as other serinekinases of interest. Alternatively, serine kinase biological activitycan be measured indirectly by measuring elevated levels of one or morephospho-proteins which are known to be phosphorylated by the kinase ofinterest. For example, as described herein, the levels of phosphorylatedtopoisomerase I was assessed by immunoprecipitation using an antibodythat binds to both the phosphorylated and unphosphorylated form of theprotein, followed by Western blotting using a phosphoserine-specificantibody. Western blots are amenable to relative quantification bydensitometric analysis.

By “phosphorylates topoisomerase I”, when referring to a serine kinaseenzyme, is meant any serine kinase enzyme which is capable of catalyzinga phosphotransferase reaction involving the transfer of the γ-phosphategroup of ATP or other nucleoside triphosphate to a serine residue of thetopoisomerase I enzyme. The capability of a serine kinase (or anyenzyme) to phosphorylate topoisomerase I can be determined using anykinase assay described herein or any other suitable assay known in theart for that particular kinase. A suitable kinase substrate representingthe serine amino acid phosphorylating site in topoisomerase I is apolypeptide of not less than 10 amino acids, having at least one aserine residue no less than four amino acid residues from eitherterminus of the polypeptide, and wherein the polypeptide is identical toa portion of the human topoisomerase I enzyme (SEQ ID NO: 3).

By “topoisomerase I inhibitor” is meant a compound that is capable ofinhibiting the DNA re-ligation enzymatic reaction catalyzed bytopoisomerase I. Preferred topoisomerase I inhibitors are capable ofcreating a stabilized DNA-topoisomerase I complex sufficient to inhibitthe enzymatic reaction. In order to determine whether a compound ofinterest is a topoisomerase I inhibitor, the relaxing of supercoiled DNAis measured in the presence of topoisomerase I and the compound ofinterest. The result is compared to an assay performed under the sameconditions in the absence of the compound of interest, wherein atopoisomerase I inhibitor reduces or prevents relaxation of thesupercoiled DNA. A suitable assay for measuring topoisomerase Iinhibition is described in the Examples contained herein. TopoisomeraseI inhibitors include, for example, plant alkaloids, plant alkaloidderivatives, camptothecin, irinotecan, topotecan, and analogs thereof,as well as non-camptothecin-derived topoisomerase I inhibitors that actsimilarly to stabilize the topoisomerase I-DNA complex.

By “stabilized complex” is meant a DNA-topoisomerase I complex in whichthe topoisomerase I catalytic activity has been partially or completelyinhibited by the further binding of a topoisomerase I inhibitor.Normally, the DNA-topoisomerase I complex is a transient chemicalintermediate species formed during the isomerase reaction. But, in thepresence of a topoisomerase I inhibitor, isomerization, DNA religation,and/or DNA release is inhibited, resulting in a stabilized complex whichinhibits DNA replication.

By “contacting”, when referring to the interaction between a cell and anagent, is meant a physical interaction between the cell (or a cellularcomponent) and the agent such that the desired biological effect isproduced as a direct or indirect result of that interaction. Contactingmay involve, for example, a physical interaction between the agent and acell surface receptor, followed by a signal transduction event resultingin the desired biological activity within the cell. Alternatively,contacting may require internalization of the agent in order for thebiological effect to be produced. Such is the case for vectors encodingserine kinase enzymes or ARF.

By a “vector” is meant a non-chromosomal nucleic acid comprising anintact replicon such that the vector may be replicated when placedwithin a cell, for example by a process of transformation, transfectionor transduction. Vectors may be viral or non-viral. Viral vectorsinclude retroviruses, adenoviruses, herpesvirus, papovirus, or otherwisemodified naturally occurring viruses. Exemplary non-viral vectors fordelivering nucleic acid include naked DNA; DNA complexed with cationiclipids, alone or in combination with cationic polymers; anionic andcationic liposomes; DNA-protein complexes and particles comprising DNAcondensed with cationic polymers such as heterogeneous polylysine,defined-length oligopeptides, and polyethylene imine, in some casescontained in liposomes; and the use of ternary complexes comprising avirus and polylysine-DNA.

By a “promoter” is meant a nucleic acid sequence sufficient to directtranscription of a gene. Also included in the invention are thosepromoter elements which are sufficient to render promoter dependent geneexpression controllable for cell type specific, tissue specific orinducible by external signals or agents (e.g. enhancers or repressors);such elements may be located in the 5′ or 3′ regions of the native gene,or within an intron.

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such a wayas to permit expression and/or secretion of the product (e.g., aprotein) of the nucleic acid molecule when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

In another aspect, the invention provides a method for inducingapoptosis, cell killing, and/or growth arrest in a cell by contactingthe cell with an agent that inhibits the binding of ARF to topoisomeraseI. The binding may be inhibited by an antibody or other binding agent(e.g. a peptide, an aptamer, or a peptidomimetic) which disrupts theinteraction between ARF to topoisomerase I. The agent may bind directlyto ARF or to topoisomerase I and may competitively or non-competitivelyinhibit the ARF-topoisomerase I binding interaction. Suitable antibodiesinclude, for example, ARF-specific antibodies and topoisomeraseI-specific antibodies. Alternatively, a phosphatase thatdephosphorylates topoisomerase I may be used to reduce ARF binding totopoisomerase I. A CK2 inhibitor such as TBB(4,5,6,7-tetrabromobenzotriazole) could also be used to reduceCK2-mediated phosphorylation of topoisomerase I. In preferredembodiments, the method disrupts existing ARF-topoisomerase I complexes.In other embodiments, ARF binding to HDM2 is increased. In otherembodiments, p53 biological activity is increased.

In another aspect, the invention provides methods for determining thesensitivity of a cancer cell to a topoisomerase I inhibitor comprising:(i) determining the nuclear localization of ARF within the cancer cell,and (ii) identifying the cancer cell as being sensitive to atopoisomerase I inhibitor when the ARF is substantially localized to thenucleolus and identifying a cancer cell as being resistant to atopoisomerase I inhibitor when said ARF is substantially disbursed inthe nucleus of said cell. In this context, the term “substantially”means greater than 50%. In preferred embodiments in which cancer cellsare identified as being sensitive to a topoisomerase inhibitor, morethan 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the ARF is present thenucleolus or the nucleolus and perinucleolar region.

In another aspect, the invention provides methods for determining thesensitivity of a cancer cell to a topoisomerase I inhibitor comprising:(i) determining the ratio of free ARF to ARF bound to topoisomerase I inthe nucleus of the cancer cell, and (ii) identifying the cancer cell asbeing sensitive to a topoisomerase I inhibitor when the ratio is lessthan 1, and identifying a cancer cell as being resistant to atopoisomerase I inhibitor when the ratio is greater than 1. In preferredembodiments in which cancer cells are identified as being sensitive to atopoisomerase inhibitor, the ratio is less than 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, or 0.1. In preferred embodiments in which cancer cellsare identified as being resistant to a topoisomerase inhibitor, theratio is greater than 2, 3, 4, 5, 7, 10, 20, 25, 50, 90, or 100.

In another aspect, the invention provides methods for determining thesensitivity of a cancer cell to a topoisomerase I inhibitor comprising:(i) determining the ratio of unphosphorylated topoisomerase I tophosphorylated topoisomerase I in the nucleus of the cancer cell, and(ii) identifying the cancer cell as being sensitive to a topoisomerase Iinhibitor when the ratio is less than 1, and identifying a cancer cellas being resistant to a topoisomerase I inhibitor when the ratio isgreater than 1. In preferred embodiments in which cancer cells areidentified as being sensitive to a topoisomerase inhibitor, the ratio isless than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. In preferredembodiments in which cancer cells are identified as being resistant to atopoisomerase inhibitor, the ratio is greater than 2, 3, 4, 5, 7, 10,20, 25, 50, 90, or 100.

In preferred embodiments of the aspects of this invention, thetopoisomerase inhibitor is camptothecin, irinotecan, or topotecan. Inother embodiments, the cancer cell is a lung cancer cell, prostatecancer cell, hepatocellular carcinoma cell, breast cancer cell,colorectal cancer cell, acute myelogenous leukemia cell, melanoma cell,or adenocarcinoma cell, ovarian cancer cell, neuroendocrine carcinomacell, gastric cancer cell, esophageal cancer cell, pancreatic cancer,adenocarcinoma, brain cancer, head and neck cancer, bone marrow-derivedcancer, bone cancer, kidney cancer, retina cancer, bladder cancer, livercancer, or mesothelioma cancer.

In another aspect, the invention provides kits for determining thesensitivity of a cell (e.g., a cancer cell) to a topoisomerase Iinhibitor. An exemplary kit may comprise (i) an anti-phosphoserineantibody specific for a serine phosphorylated epitope on topoisomeraseI, and (ii) an anti-topoisomerase I antibody. The kit may also include(iii) an anti-ARF antibody. In a preferred embodiment, theanti-topoisomerase I antibody binds to human topoisomerase I. In anotherpreferred embodiment, the anti-ARF antibody binds to human ARF.

In another aspect, the invention provides a cell containing arecombinant vector and a topoisomerase I inhibitor. Suitable recombinantvectors include vectors encoding a serine kinase (e.g., CKII or PKC),ARF, or a biologically active fragment of ARF. In preferred embodiments,the cell further contains a stabilized DNA-topoisomerase I complex.

In another aspect, the invention provides a cell comprising atopoisomerase I inhibitor and further expressing an elevated serinekinase biological activity, wherein the cell has been contacted with anagent that elevates the serine kinase biological activity relative tothe serine kinase biological activity in the same cell which has notbeen contacted with the agent.

In another aspect, the invention provides methods for determining thesensitivity of a cancer cell to a topoisomerase I inhibitor, comprising:(i) determining status of phosphorylation on serine 506 amino acidresidue of topoisomerase I within the cancer cell by way of an assay;and (ii) identifying the cancer cell as being sensitive to thetopoisomerase I inhibitor when phosphorylation of serine 506 amino acidresidue of topoisomerase I is above a predetermined threshold asdetermined by the assay, and identifying said cancer cell as beingresistant to the topoisomerase I inhibitor when phosphorylation ofserine 506 amino acid residue of topoisomerase I is below thepredetermined threshold as determined by the assay. In preferredembodiments, the predetermined threshold is a ratio of unphosphorylatedtopoisomerase Ito phosphorylated topoisomerase I within said cancercell. In some embodiment, CKII RNA expression of the cancer cell can beevaluated as a confirmatory or supportive diagnostic test.

In preferred embodiments of the aspects of this invention, the presenceor absence of phosphorylation on serine 506 amino acid residue oftopoisomerase I is determined by an antibody based assay, which mayinclude an antibody that binds phosphorylated serine 506 amino acidresidue of topoisomerase I, but does not bind nonphosphorylated serine506 amino acid residue of topoisomerase I, and or include an antibodythat binds unphosphorylated serine 506 amino acid residue oftopoisomerase I, but does not bind phosphorylated serine 506 amino acidresidue of topoisomerase I. In some embodiments the antibody may be amonoclonal antibody, and in some embodiments the antibody may be apolyclonal antibody.

In another aspect, the invention provides methods for treating cancer ina patient, comprising: (i) determining status of phosphorylation, by wayof an assay, on serine 506 amino acid residue of topoisomerase I in abiological specimen from the patient; (ii) identifying the patient asbeing sensitive to a topoisomerase I inhibitor when phosphorylation ofserine 506 amino acid residue of topoisomerase I is above apredetermined threshold as determined by the assay; and (iii)administering the topoisomerase I inhibitor to the patient. In someembodiments, the biological specimen may be tumor cells, tumor tissue,blood, urine, and/or sputum.

In another aspect, the invention provides methods of increasing thesensitivity of a cancer patient to a topoisomerase inhibitor, comprisingadministering a CKII activator to the cancer patient in an amountsufficient to decrease ratio of unphosphorylated topoisomerase I tophosphorylated topoisomerase I. In some embodiments, the CKII activatoris 1-ethyl-4,5-dicarbamoylimidazole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of electrophoretic gel separations of nuclearproteins from DU145, H358, and H23 cells. FIG. 1A is a silver-stainedelectrophoretic gel showing cellular proteins corresponding in size totopoisomerase I that bound to an immobilized fusion protein composed ofthe N-terminal portion of ARF (ARF-N-term; exon 1β, amino acid residues1-64) and the full-length ARF protein. FIG. 1B shows a Western analysisof topoisomerase I that bound to immobilized full length ARF orNickel-NTA agarose lacking immobilized fusion protein (control, middlepanel). FIG. 1C shows co-binding of topoisomerase I and ARF followingimmunoprecipitation of H358 and H23 nuclear extracts with an anti-topo Iantibody followed by Western detection of topo I or ARF in theimmunoprecipitated material, before (left panel) or 48 hours aftertreatment with Adp14 (replication-defective adenoviral vector encodingfull length ARF), moi=20 pfu/cell (middle panel). Far right panel showsmaterial that remained unbound by the anti-topo I antibody.

FIG. 2A is a graph showing that nuclear extracts from H358 cells (closedcircles) have greater topoisomerase I activity compared to and H23 cells(open circles) in an in vitro assay measuring the conversion ofsupercoiled plasmid DNA (“s”) to the relaxed form (“r”). FIG. 2B is anagarose gel electrophoresis of reaction products of a typical in vitrotopoisomerase I assay in which 0.32, 0.65, or 1.3 μg of H358 extract(lanes 1-3, respectively) or H23 extract (lanes 4-6, respectively) wereadded per reaction. Lane “C” (control) shows migration of supercoiledplasmid in the absence of cell extract. FIG. 2C is an agarose gelelectrophoresis showing that addition of purified ARF bacterial fusionprotein in the topoisomerase activity assay increases the topoisomeraseI activity of H358 nuclear extracts (lanes 1-3), but not H23 nuclearextracts (lanes 4-6). The purified ARF-N-term bacterial fusion protein,which retains only the N-terminal 64 amino acid residues of ARF (lanes7-12) had no effect on topoisomerase I activity.

FIG. 3A is an electrophoretic gel showing that ARF binds totopoisomerase I in H358 nuclear extracts, but not in H23 nuclearextracts. The ARF-topoisomerase I complexes are destroyed by alkalinephosphatase (+AP) treatment and restored in both cell types followingCKII treatment. FIG. 3C shows that this effect is also achieved usingpurified topoisomerase I. FIG. 3C also shows that HT29 cells have lowlevels of topo I serine phosphorylation and ARF-topoisomerase Icomplexation relative to H358 cells. This data demonstrates that theARF-topoisomerase I complex formation is a phosphorylation dependentevent. FIG. 3B is an electrophoretic gel showing that the catalyticactivity of topoisomerase I in H358 cells is abolished by alkalinephosphatase treatment and the activity cannot be enhanced byoverexpression of ARF. FIG. 3D is a bar graph showing the CKII activityin lysates of H358, H23, and HT29 cells.

FIG. 4A is an electrophoretic gel separation and Western oftopoisomerase I and ARF following subcellular fractionation. These datashow that topoisomerase I is concentrated in the nucleolus of both H538and H23 cells, and ARF is also concentrated in the nucleolus of H538cells. By contrast, ARF has is distributed approximately evenly betweenthe nucleolus and the nucleoplasm of H23 cells. FIG. 4B is a series ofphotomicrographs showing the immunofluorescence pattern of ARF in fixedand permeabilized H358 and H23 cells. This confirms the findings of FIG.4A and demonstrates that there is reduced nucleolar ARF localization inH23 cells. FIG. 4C is an electrophoretic separation followingco-immunoprecipitation analysis of Nucleophosmin (NPM/B23) and ARF inH358 and H23 nuclear extracts.

FIG. 5A is an electrophoretic separation and Western analysis of H358cellular actin (top row) or ARF (bottom row) 48 hours after treatmentwith Adp14 (replication-defective adenoviral vector encoding full-lengthARF) (lane 1) or Ad1β (replication-defective adenoviral vector encodingthe N-terminal 64 amino acid residues of ARF encoding by the first exon(exon 1β of ARF) (lane 2), or 72 hours after treatment with siRNAcontrol sequence (lane 3), or ARF siRNA to exon 2 (lane 5). Lane 4 showsactin and ARF levels in untreated H358 cells. Digital analyses of ARFband intensities are shown beneath the ARF Western blot. FIG. 5B is aseries of graphs showing H358 and PC-3 cell viabilities assayed 5 dayspost-vector treatment (adenoviral vector, moi 20 pfu/cell, or siRNA),and 4 days post treatment with increasing doses of camptothecin.Viability is expressed as a percent of no-camptothecin control for eachvector or siRNA treatment. Results represent average of triplicatewells, with standard deviations indicated. Treatments: Adp14 (ARF fulllength ▪); Ad1β (ARF N-term ◯); siRNA control (▾); no vector (); siRNAexon 2 (∇). FIG. 5C shows the results of a Western analysis (top panel)of H23 cellular actin and ARF levels in untreated cells (lane 1) or 72hours after treatment with ARF siRNA to exon 1β (lane 3), or siRNA plusAdp14 (moi=100) (lane 2). Digital analyses of ARF levels are shown belowARF lanes. (lower panel) H358 cell viability assay following theindicated treatments. Viability was measured 3 days post-start ofcamptothecin treatment. Together, these data demonstrate that reducedARF levels, and thus reduced ARF-topoisomerase I complex formation (seeFIG. 5D), renders cells less sensitive to topoisomerase I inhibitors.

FIG. 5D shows ARF-topoisomerase I complex formation in H358 cellsfollowing various treatments, and correlates differences in complexformation with differences in topoisomerase I activity. FIG. 5D (upperpanel shows topoisomerase I immunoprecipitation followed bytopoisomerase I or ARF Western following various treatments. Lanescorrespond to the same treatments as in FIG. 5A. Digital analyses of ARFband intensities are shown below the ARF lanes. FIG. 5D (middle panel)is an ethidium bromide-stained agarose gel of the reaction products ofan in vitro topoisomerase I assay measuring loss of supercoiled plasmidDNA in the presence of 0.06 μg H358 nuclear extract (amount thatconverts 50% of supercoiled plasmid to relaxed form; see FIG. 2A).Numbered lanes correspond to the same treatments as in FIG. 5A.s=supercoiled; r=relaxed form. FIG. 5D (lower panel) is a graphicalrepresentation of the relative supercoil band intensities of lanes 1-5of the ethidium bromide-stained agarose gel shown in the middle panel.These data demonstrate that ARF-topoisomerase I complex formation andtopoisomerase I activity were altered in a predictable and coordinatemanner by overexpressing or inhibiting ARF. FIG. 5E is a graph showingthe H23 cell viability assay performed as described above. Consistentwith the observation that topoisomerase I activity in H23 cells is notenhanced by ARF overexpression, this experiment demonstrates that ARFoverexpression does not render H23 cells sensitive to topoisomerase Iinhibitors.

FIG. 6 is a series of electrophoretic gels showing that ARF bindingpromotes topoisomerase I complex formation with DNA. The top panel showsthe results of an immunodepletion assay carried out on nuclei preparedfrom cells treated with increasing doses of Adp14, followed bycamptothecin treatment to crosslink topoisomerase I onto DNA. The gelshows that increasing levels of ARF lead to a reduction in the bandintensity of topoisomerase I, indicating that more topoisomerase I hasbecome covalently bound to DNA by camptothecin and therefore cannotenter the gel. The middle and bottom panels show topoisomerase Iimmunoprecipitation followed by an ARF and a topoisomerase I Westernanalysis, respectively, in cells treated with increasing doses of Adp14.Digital analyses of topoisomerase I and ARF levels are shown belowlanes. The results show that increasing doses of Adp14 promoteincreasing levels of ARF-topoisomerase I complex formation, and thatthis promotes increased topoisomerase I binding to DNA followingcamptothecin treatment.

FIG. 7 is a series of graphs showing the correlation of serinephosphorylation, ARF/topoisomerase I complex formation and camptothecinsensitivity. FIGS. 7A-C show the relative amounts of (A) serinephosphorylation of topoisomerase I, (B) total topoisomerase I, and (C)ARF-topoisomerase I complex following topoisomerase Iimmunoprecipitation in the indicated cell types. FIG. 7D is a graphshowing the viability of the indicated cell types 3 days after treatmentwith camptothecin.

FIG. 8A is a graph showing the viability of the indicated cell types 3days after treatment with camptothecin. FIG. 8B is an electrophoreticgel of a topoisomerase I immunoprecipitation followed by an ARF andtopoisomerase I Western analysis. FIG. 8C is an electrophoretic gel of atopoisomerase I immunoprecipiation and a phosphoserine Western analysis.FIG. 8D is an ARF Western analysis. Together, these data demonstratethat the level of ARF-topoisomerase I complex formation in Hela cells isintermediate to that of H538 and H23 cells. The sensitivity of Helacells to topoisomerase I inhibitors is also intermediate to that of H538and H23 cells. The fact that Hela cell topoisomerase I is serinephosphorylated indicates that abnormalities other than serinephosphorylation can disrupt the ARF/topoisomerase I complex and promoteresistance to camptothecin.

FIG. 9 is the amino acid sequence of human ARF, as provided in accessionno. NP_(—)478102 (SEQ ID NO: 2).

FIG. 10 is the amino acid sequence of human topoisomerase I, as providedin accession no NM_(—)003286. (SEQ ID NO: 3).

FIG. 11A shows a graph of 3-day viability assays carried out in 96 wellplates. FIG. 11B1-B3 show bar graphs of PKC, CK2, and cdk1 enzyme levelsand Western blots of PKC, CK2, and cdk1 protein levels in cancer celllines. Western blots of actin levels serves as a control. FIG. 11C1-C3show bar graphs and Western blots of PKC, CK2, and cdk1 activities andprotein levels, respectively, in normal cell lines, compared to H358 andH23. FIG. 11D shows topo-I immunoprecipitation followed by topo-IWestern (top row) or phosphoserine Western (bottom row).

FIG. 12 shows the result of a semi quantitative PCR of CK2 mRNA levels.Analysis of CK2 mRNA levels in cellular RNA, normalized to levels inHET1A cells, showed that levels in normal cells (HET1A, BJ-1, GT41F) andthe 3 camptothecin-resistant cancer cell lines (H23, HT29, SW480) arelower than levels in the 6 camptothecin-sensitive cancer cell lines(H358, PC3, DU145, LnCAP, MDAMB-435, OC3). Digital quantitation of bandintensities for CK2 are shown below the lanes.

FIG. 13A shows a graph of 3-day viability assay ofcamptothecin-sensitive H358 lung cancer cells, pretreated with the CK2inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB) (10 μM, 1 hr) ortransfected with CK2 siRNA, or control, scrambled sequence siRNA,followed immediately by treatment with increasing doses of camptothecinfor 18 hours. Control cells received no pretreatment. FIG. 13B shows agraph of 3-day viability assay of camptothecin-resistant H23 cells,treated for 18 hours with increasing doses of camptothecin, in thepresence or absence of the CK2 activator,1-ethyl-4,5-dicarbamoylimidazole. CK2 activator treatment was for theduration of the assay. Cell viability was scored 3 days post-start ofcamptothecin treatment.

FIG. 14A shows a Western analysis of recombinant, baculovirus-expressedhuman topo I, dephosphorylated with alkaline phosphatase (+AP) ordephosphorylated with AP followed by rephosphorylation with CK2(+AP+CK2), using the topo I ser506 phosphospecific antibody (top), andan antibody to total topo I (bottom). FIG. 14B shows a Western analysisof cellular topo I from H358 and H23 non small cell lung cancer cellsusing the topo I ser506 phosphospecific antibody (top), an antibody tototal topo I (middle), and a control antibody to the non phosphorylatedtopo I ser 506 site (bottom). FIG. 14C shows a Western analysis ofcellular topo I from a series of human cancer cell lines and normalcells (BJ-1 and HET1A) using the topo I ser506 phosphospecific antibody(top), and an antibody to total topo I (bottom). FIG. 14D showsimmunofluorescence staining of H358 cells with the topo I ser506phosphospecific antibody (1:100). Cells were fixed and permeabilized asdescribed in Lee, et al., Cancer Research 2005; 65:9834-9842, followedby 2 minute denaturation with 0.5% SDS. Secondary antibody wasanti-rabbit IgG Alexafluor 486 (Molecular Probes, Inc.). Cancer celllines are as follows: H358 non small cell lung cancer, PC-3 prostatecancer, DU145 prostate cancer, H23 non small cell lung cancer, HT29colon cancer, LnCAP prostate cancer, OC3 esophageal cancer, MB435 breastcancer (abbreviated from MDA MB 435). FIG. 14E shows a Western analysesof phospho topoisomerase I (using the topo I ser506 phosphospecificantibody, top), and total topoisomerase I (bottom) in lysates ofimmortalized normal cell lines BJ1 (human fibroblasts) and HET1A (humanepithelial cells). FIG. 14F shows a Western analysis of phosphotopoisomerase I (using the topo I ser506 phosphospecific antibody, top)and total topoisomerase I (bottom) in lysates of H23 cells with orwithout treatment with the CK2 activator as in FIG. 13B, and in lysatesof H358 cells with or without treatment with the CK2 inhibitor, TBB, asin FIG. 13A.

FIG. 15A shows a bar graph of assays of CK2 activity in nuclear extractsprepared from H358 cells (lanes 1-4) and H23 cells (lanes 5,6) 3 daysafter the following treatments: treated as follows: 1—no CK2manipulation; 2—transfection with control, scrambled siRNA; 3—treatment1 hour with 10 μM TBB; 4—transfection with CK2 siRNA; 5—treatment (forduration of assay) with CK2 activator; 6—no CK2 manipulation. Each assayrepresents the average of duplicate samples, with standard deviationsshown. FIG. 15B shows Western analysis of CK2 or actin (control) proteinin lysates of H358 or H23 cells, 3 days after treatments in FIG. 15A.FIG. 15C shows immunoprecipitations with anti-topo I of nuclear lysatesof H358 or H23 cells, 3 days after treatments in FIG. 15A, followed byWestern analysis of topo I (top row), phosphoserine (middle row), orp14ARF (bottom row) in the immunoprecipitated material. FIG. 15D showsenzymatic assays of topoisomerase I-mediated conversion of supercoiledplasmid (S) to relaxed form (r) by nuclear extracts prepared from H358or H23 cells 3 days after treatments in FIG. 15A. P=supercoiled plasmidonly. FIG. 15E shows bar graph of K+/SDS precipitation of covalent,camptothecin-stabilized cleavage complexes between topo I and cellularDNA of H358 or H23 cells. Cells were treated as in FIG. 15A, followed 3days later by overnight labeling with [3H]-thymidine, followed by a 25minute incubation in 0.08 μM camptothecin. FIG. 15F shows Westernanalysis of γ-H2A.X and total H2A.X (control) in nuclear lysates of H358and H23 cells, 3 days after the treatments in FIG. 15A. FIG. 15G showsbar graph of purified CK2 holoenzyme (Promega, Madison, Wis.), CK2α1catalytic subunit (Active Motif, Carlsbad, Calif.), PKC (Sigma, St.Louis, Mo.), and cdk1 (Enzo Life Sciences, Farmingdale, N.Y.) wereassayed in vitro using assay kits purchased from UpsateBiotechnology/Millipore, Temecula, Calif.) in the absence (−) orpresence (+) of 10 nM CK2 activator, 1-ethyl-4,5-dicarbamoylimidazole. 2units of each enzyme were assayed. FIG. 15H shows a bar graph where H23cells were left untreated (−) or treated (+) with 10 nM CK2 activator,without changing the medium during the three day assay. Cell lysateswere then prepared and assayed for PKC and cdk1 activity as described inMaterials and Methods. FIG. 15I shows H23 cells were left untreated (−)or exposed to 10 nM CK2 activator (+). RNA was isolated 3 days laterusing an RNA isolation kit (Qiagen, Valencia, Calif.) and RT-PCR wasperformed using CK2α primers and conditions described in Kramerov etal., Am J Pathol, 2006; 168:1722-1736, which produce a 151 bp DNAfragment revealed by ethidium bromide-stained agarose gelelectrophoresis. Actin amplification of parallel aliquots served as acontrol. The results show that the CK2 activator did not act at thelevel of CK2α transcription.

FIG. 16 shows a diagram summarizing an embodiment of how CK2 activatorsand/or ARF can be used to enhance camptothecin (CPT) sensitivity.

DETAILED DESCRIPTION OF INVENTION

The present inventions are based on different mechanisms for inducingcell death, apoptosis and/or growth arrest in cancer cells. Eachmechanism is based upon altering (i.e., increasing or decreasing) theamount of ARF-topoisomerase I complex formation. One mechanism is basedon the discovery that reduced topoisomerase I serine phosphorylationand/or ARF-topoisomerase I complex formation renders cells lesssensitive (or insensitive) to the apoptotic and/or growth arrestingeffects of topoisomerase I inhibitors. Sensitivity to topoisomerase Iinhibitors may be restored by increasing amount of ARF-topoisomerase Icomplex formation which may be done by increasing the serinephosphorylation of the enzyme (e.g., using CKII or PKC), and/or byincreasing ARF in order to promote complex formation. Another mechanismis based on the discovery that disruption of ARF-topoisomerase I complexformation correlates with cell death, apoptosis and/or growth arrest.

As described in more detail in the following examples, analysis of theH23 non-small cell cancer cell line identified cancer-related defects intopoisomerase I-ARF binding. Specifically, the loss of topoisomerase Iserine phosphorylation caused a corresponding loss of topoisomerase Iactivity. Additionally, the absence of topoisomerase I serinephosphorylation resulted in reduced ARF binding and caused an aberrantnuclear distribution of ARF. It was further observed in H23 cells thatonly about half of the cellular ARF was bound to NPM, a nucleolarprotein. Normally, virtually all cellular ARF is NPM-bound.

Increased ARF-Topoisomerase I Complex Formation Increases Sensitivity toTopoisomerase I Inhibitors.

The mechanisms that regulate topoisomerase I activity are ofconsiderable therapeutic interest, since topoisomerase I has proven tobe an important target for chemotherapy (Pommier, et al., BiochimBiophys Acta 1998; 1400(1-3):83-105; Liu, L. F., Annu Rev Biochem 1989;58:351-75). A potent class of chemotherapeutic drugs that targettopoisomerase I are derived from the plant alkaloid, camptothecin, agroup that includes irinotecan (Camptosar) and Topotecan. These agentshave been highly effective for the treatment of a variety of solidtumors that have shown resistance to other treatments, includingnon-small cell lung cancer (Rothenberg, M. L., Oncologist 2001;6(1):66-80). Camptothecin and its derivatives prevent the re-ligation ofthe cleavable complex, a topoisomerase I reaction intermediate, therebycreating lethal topoisomerase I-induced DNA strand breaks (Champoux, J.J., Annu Rev Biochem 2001; 70:369-413). In addition, severalnon-camptothecin-derived topoisomerase I inhibitors that act through asimilar mechanism are being developed and evaluated (Pommier, ChemicalReviews 2009; 109:2894-2902). As with many chemotherapeutic treatments,however, de novo or acquired resistance to camptothecins is common, andcan occur through a variety of mechanisms (Rasheed, et al., Oncogene2003; 22(47):7296-304; Xu, et al., Ann Oncol 2002; 13(12):1841-51),including downregulation of topoisomerase I activity (Pommier, et al.,Ann N Y Acad Sci 1996; 803:60-73).

The following examples demonstrate that reduced levels of topoisomeraseI activity and failure of ARF/topoisomerase I complex formation in H23cells correlates with camptothecin resistance, while ectopic overexpression of ARF and increased ARF/topoisomerase I complex formation inH358 cells results in enhanced camptothecin sensitivity (FIG. 5).

Without wishing to be bound by any theory, it is believed that theapoptosis, cell killing and/or growth arrest caused by topoisomerase Iinhibitors requires a catalytically active topoisomerase I enzyme.Catalytic activity is enhanced by ARF-topoisomerase I complex formation,which itself requires serine phosphorylation of the enzyme. Thus, thefollowing examples demonstrate that ARF-topoisomerase I complexformation can be increased by increasing the amount of serinephosphorylation of the enzyme and/or increasing the amount of ARF (or abiologically active fragment of ARF) available for topoisomerase Ibinding. The resulting elevation in ARF-topoisomerase I complexformation increases the sensitivity of the cell to topoisomerase Iinhibitors which bind to, and stabilize, the covalent complex formed asan intermediate during the isomerase reaction. The stabilized complexeslikely prevent further DNA replication.

Disruption of ARF-Topoisomerase I Complex Formation Induces Apoptosisand/or Growth Arrest in Cancer Cells.

ARF is a well known positive regulator of the p53 tumor suppressor. ARFinteracts with and sequesters human double minute (HDM2) or itsequivalent, a negative regulator of p53. In doing so, ARF promotes theaccumulation of p53 protein which results in p53-mediated cell cyclearrest or apoptosis.

As demonstrated herein, ARF is normally localized to the nucleolus as aresult of its topoisomerase I binding. This effectively prevents ARFfrom binding to HDM2, thereby permitting HDM2-inhibition of p53.However, disruption of the ARF-topoisomerase I binding interactionallows ARF to redistribute from the nucleolus to the nucleoplasm (FIG.4). Without wishing to be bound by any theory, it is believed that thisredistribution allows ARF to bind and sequester HDM2, causing adis-inhibition or an activation of p53. It is this p53 activation whichunderlies the apoptotic and growth arresting effect caused by thedisruption of ARF-topoisomerase I complex formation.

Vectors Suitable for Delivery to Humans

This invention features methods and compositions for treating cancer.The cancer may be treated by inducing cell death (e.g., apoptosis) orgrowth arrest in the cancer cells. In one aspect, the invention featuresmethods of gene therapy to express ARF or a serine kinase (e.g., CKII orPKC) in the cancer cells of a patient. Gene therapy, including the useof viral vectors as described herein, seeks to transfer new geneticmaterial (e.g., polynucleotides encoding a serine kinase) to the cellsof a patient with resulting therapeutic benefit to the patient. For invivo gene therapy, expression vectors encoding the gene of interest isadministered directly to the patient. The vectors are taken up by thetarget cells and the serine kinase gene expressed. Several recentreviews are available discussing methods and compositions for use ingene therapy (Eck et al., in Goodman & Gilman's The PharmacologicalBasis of Therapeutics, Ninth Edition, Hardman et al., eds., McGray-Hill,New York, 1996, Chapter 5, pp. 77-101; Wilson, Clin. Exp. Immunol. 107(Suppl. 1):31-32, 1997; Wivel et al., Hematology/Oncology Clinics ofNorth America, Gene Therapy, S. L. Eck, ed., 12(3):483-501, 1998; Romanoet al., Stem Cells, 18:19-39, 2000, U.S. Pat. No. 6,080,728).

Adenoviruses are able to transfect a wide variety of cell types,including non-dividing cells. There are more than 50 serotypes ofadenoviruses that are known in the art, but the most commonly usedserotypes for gene therapy are type 2 and type 5. Typically, theseviruses are replication-defective; genetically modified to preventunintended spread of the virus. This is normally achieved through thedeletion of the E1 region, deletion of the E1 region along with deletionof either the E2 or E4 region, or deletion of the entire adenovirusgenome except the cis-acting inverted terminal repeats and a packagingsignal (Gardlik et al., Med. Sci. Monit. 11: RAI 10-121, 2005).

Retroviruses are also useful as gene therapy vectors and usually (withthe exception of lentiviruses) are not capable of transfectingnon-dividing cells. The invention includes use of any appropriate typeof retrovirus that is known in the art, including, but not limited to,HIV, SIV, FIV, EIAV, and Moloney Murine Leukaemia Virus (MoMLV).Typically, therapeutically useful retroviruses including deletions ofthe gag, pol, or env genes.

Adeno-associated virus (AAV) vectors can achieve latent infection of abroad range of cell types, exhibiting the desired characteristic ofpersistent expression of a therapeutic gene in a patient. The inventionincludes the use of any appropriate type of adeno-associated virus knownin the art including, but not limited to AAV1, AAV2, AAV3, AAV4, AAV5,and AAV6 (Lee et al., Biochem J. 387: 1-15, 2005; U.S. PatentPublication 2006/0204519).

Exemplary non-viral vectors for delivering nucleic acid include nakedDNA; DNA complexed with cationic lipids, alone or in combination withcationic polymers; anionic and cationic liposomes; DNA-protein complexesand particles comprising DNA condensed with cationic polymers such asheterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, in some cases contained in liposomes; and the use of ternarycomplexes comprising a virus and polylysine-DNA. In vivo DNA-mediatedgene transfer into a variety of different target sites has been studiedextensively. Naked DNA may be administered using an injection, a genegun, or electroporation. Naked DNA can provide long-term expression inmuscle (Wolff, et al., Human Mol. Genet., 1:363-369, 1992; Wolff, etal., Science, 247, 1465-1468, 1990). DNA-mediated gene transfer has alsobeen characterized in liver, heart, lung, brain and endothelial cells(Zhu, et al., Science, 261: 209-211, 1993; Nabel, et al., Science,244:1342-1344, 1989). DNA for gene transfer also may be used inassociation with various cationic lipids, polycations and otherconjugating substances (Przybylska et al., J. Gene Med., 6: 85-92, 2004;Svahn, et al., J. Gene Med., 6: S36-S44, 2004).

Methods of gene therapy using cationic liposomes are also well known inthe art. Exemplary cationic liposomes for use in this invention areDOTMA, DOPE, DOSPA, DOTAP, DC-Chol, Lipid GL-67™, and EDMPC. Theseliposomes may be used to encapsulate a serine kinase vector for deliveryinto target cells.

Typically, vectors made in accordance with the principles of thisdisclosure will contain promoters that will cause constitutiveexpression of the serine kinase coding sequence, although induciblepromoters may be used.

Administration of Topoisomerase I Inhibitors

In addition to elevating the serine kinase levels (e.g., CKII and PKClevels) in a cancer cell, sufficient to increase phosphorylation oftopoisomerase I, or increasing ARF levels sufficient to enhance theformation of an ARF/topoisomerase I complex it is desirable that thecancer cells be further contacted with one or more topoisomerase Iinhibitors. Typically, patients diagnosed as having cancer will beadministered a pharmaceutical formulation containing a topoisomerase Iinhibitor. Suitable topoisomerase I inhibitors include, for example,camptothecin, irinotecan, topotecan, and analogs of these inhibitors, aswell as non-camptothecin-derived topoisomerase I inhibitors that actsimilarly to stabilize the topoisomerase I-DNA complex. Theadministration of topoisomerase I inhibitors may be by any suitablemeans that results in an anti-neoplastic effect. The topoisomerase Iinhibitor may be administered in any appropriate amount, in any suitablecarrier substance, and is generally present in an amount of 1-95% byweight of the total weight of the composition. The composition may beprovided in a dosage form that is suitable for the oral, parenteral(e.g., intravenously, intramuscularly), rectal, or transdermaladministration. Thus, the composition may be in form of, e.g., tablets,capsules, pills, powders, granulates, suspensions, emulsions, solutions,gels including hydrogels, pastes, ointments, creams, suppositories,enemas, or injectables. The pharmaceutical compositions may beformulated according to conventional pharmaceutical practice (see, e.g.,Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A. R.Gennaro, 1995, Mack Publishing Company, Easton, Pa. and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 19881999, Marcel Dekker, New York.

Camptothecin, irinotecan, topotecan and their analogs, for example, maybe administered at doses of about 0.1-1000 mg/kg/day (e.g., about 1, 10,25, 50, 75, 100, 250, 500, 750, or 1000 mg/kg/day) (U.S. Pat. Nos.5,004,758, 5,340,817, 5,633,016, 5,859,022, 5,910,491, 6,040,306,6,214,821, 6,534,080; hereby incorporated by reference), or over therecommended dose rage of 50 to 350 mg/m² to patients, in accordance withdosing schedules recommended by the drug manufacturer. Administration ofany of the topoisomerase I inhibitors described herein may continue forabout a week, a month, six months, a year, or even the lifetime of thepatient.

Protein Kinase CK2 as a Central Regulator of Topoisomerase IHyperphosphorylation and Activity and Cellular Sensitivity toCamptothecin.

Experimental inhibition or activation of CK2 demonstrates that CK2 isnecessary and sufficient for regulating these properties oftopoisomerase I and for altering cancer cell responses to camptothecin.The results establish a cause and effect relationship between CK2activation and camptothecin sensitivity. Biomarkers based on CK2,topoisomerase I phosphorylation, or topoisomerase I/p14ARF complexformation can provide diagnostic indicators of therapy responsivetumors.

The present inventors have reported on two non small cell lung cancercell lines, H358 and H23, that express similar levels of topo I proteinbut have high and low sensitivity to camptothecin, respectively, thatcorrelates with high or low levels of topo I serine phosphorylation andtopo I activity (Bandyopadhyay, et. al., Biochemistry 2007;46:14325-14334). They have also found that the underphosphorylated andless active form of topo I in H23 cells can be activated by CK2treatment in vitro, further suggesting that CK2 could be a factor invivo in regulating camptothecin sensitivity in cells. Taken together,these observations suggest that one or more topo I serinephosphorylating activities could have a general role in a variety ofcancers to regulate topo I activity in vivo in ways that affect thecellular response to camptothecin-related drugs.

As demonstrated herein, CK2 is frequently upregulated in cancer celllines, and that levels of CK2, unlike PKC and cdk-1, display consistentcorrelation with the appearance of hyperphosphorylated topo I and withincreased cellular sensitivity to camptothecin. Furthermore,experimental modulation of cellular CK2 activity demonstrate afunctional relationship between CK2 overexpression, topo Ihyperphosphorylation, and cellular sensitivity to camptothecin. Theseresults identify CK2 as a frequent and central regulator of cellularsensitivity to camptothecin in cancer cell lines. Thus, biomarkers basedon CK2, topoisomerase I phosphorylation, or topoisomerase I/p14ARFcomplex formation can provide diagnostic indicators of therapyresponsive tumors.

Camptothecin Sensitivity of Normal and Cancer-Derived Cell LinesCorrelates with Topo I Phosphorylation and CK2 Activity but not PKC orcdk1 Activity.

Cell lines with overexpressed CK2 (FIGS. 11B1-B3 and 11C1-C3) displayhyper serine phosphorylation of topo I (FIG. 11D) that correlate withsensitivity to camptothecin (FIG. 1A). The cellular levels of two otherserine kinases, PKC and cycline-dependent kinase 1(cdk1), both of whichhave been implicated in topo I serine phosphorylation, do not correlatewith sensitivity to camptothecin (FIG. 11A-C3).

Furthermore, camptothecin sensitivity of normal and cancer-derived celllines correlate with CK2 mRNA levels, indicating that a PCR-based assayto measure CK2 mRNA levels can be used clinically to identify tumorsresponsive to camptothecin and related drugs. As shown in FIG. 12, asemi-quantitative RT-PCR analysis of CK2 mRNA levels in cellular RNA,normalized to levels in HET1 A cells, showed that levels in normal cells(HET1A, BJ-1, GT41F) and the 3 camptothecin-resistant cancer cell lines(H23, HT29, SW480) are lower than levels in the 6 camptothecin-sensitivecancer cell lines (H358, PC3, DU145, LnCAP, MDAMB-435, OC3).

In addition, a functional relationship has been established between CK2and the cellular response to camptothecin (FIGS. 13A and B), furthervalidating CK2 as a biomarker for therapy responsiveness. Experimentalinhibition of CK2 in camptothecin-sensitive H358 cells makes these cellsmore resistant to camptothecin (FIG. 13A), and conversely, experimentalactivation of CK2 in camptothecin-resistant H23 cells makes them moresensitive to camptothecin (FIG. 13B). These experiments show that CK2 isnecessary and sufficient for inducing the hyperphosphorylation andactivation of topo I that leads to increase cellular sensitivity tocamptothecin.

Novel Topoisomerase I Phospho Epitope Identifies Camptothecin-SensitiveCancer Cell Lines.

A novel CK2-mediated phosphorylation site on serine position 506 of topoI has been identified, which correlates with tumor cell sensitivity tocamptothecin, a topo I drug from which a potent class ofchemotherapeutic agents have been derived, including irinotecan andtopotecan.

As demonstrated herein, the phospho-specific IgG is immunoreactive withcellular topo I from the camptothecin-sensitive H358 cell line, but notwith cellular topo I from the camptothecin-resistant H23 cell line,following Western analysis of cell lysates (FIG. 14B), producing, in thecase of H358, a single immunoreactive band. In contrast, the control,non-phospho-specific IgG is poorly immunoreactive with H383 topo I, butstrongly immunoreactive with H23 topo I, following Western analysis ofcell lysates (FIG. 14B). The phospho-specific antibody lacksimmunoreactivity with topo I from the immortalized normal cell lines,BJ-1 and HET1A, indicating that the phosphorylated epitope is absentfrom these normal cells.

A functional relationship between CK2 activation and the expression ofthe phosphoserine 506 epitope was demonstrated by showing thatexperimental activation of CK2 in H23 cells treated with the CK2activator (1-ethyl-4,5-dicarbamoylimidazole) induces cellular levels ofthe phosphoserine 506 epitope (FIG. 14E), and experimental inhibition ofCK2 in H358 cells treated with tetrabromobenzotriazole (TBB) suppressescellular levels of the phosphoserine 506 epitope (FIG. 14E).

The phosphospecific IgG is immunoreactive with endogenous topo I infixed, permeabilized H358 cells, as shown by the immunofluorescenceimage in FIG. 14D, indicating that it can be employed in multipleformats to detect the phosphorylated epitope. Taken together, theresults support a model in which abnormal CK2-mediated phosphorylationof topo I on serine 506 in cancer cells expressing elevated CK2 activityleads to elevated topo I activity and increased cellular sensitivity tocamptothecin.

The phosphorylated serine 506 site appears to be cancer specific,suggesting additional applications for early diagnosis. An earlierpublished study identified topo I serine phosphorylation sites at aminoacid positions 10, 21, 112, and 394, mediated by either CK2 (ser 10),PKC (ser 21) or cdk1 (ser 112 and 394) (Hackbarth, et al., J Biol Chem2008; 283:16711-16722). The present inventors have observed that theA549 and K562 cancer cell lines used to identify these other serinephosphorylation sites, display low levels of topo I phosphorylation andactivity similar to levels observed in the poorly phosphorylated H23cancer cell line, and in the normal cell lines GT41F, BJ-1, and HET1A.Furthermore, the Hackbarth, et al. study found that the phosphorylatedprotein was some two-fold more active than the dephosphorylated protein,and that the effect was mediated entirely by phosphorylation at position21, a PKC site. Thus, the CK2-targeted site that the present inventorshave identified at position 506 is distinct from the previouslyidentified sites, and is likely to represent an aberrant phosphorylationevent characteristic of cancer cells that overexpress CK2.

The topo-I phosphorylation on serine 506 appears to be a common minimalrequirement for camptothecin sensitivity, and that cells that lack thisphosphorylation site will be resistant to camptothecin. Therefore, adiagnostic assay to detect this phosphoserine epitope can distinguishpatients likely to respond to camptothecin-based therapies from patientsunlikely to respond, and can guide physicians in the choice of treatmentstrategies.

The serine 506 epitope of topo I can be exploited for the development ofassays, such as immunoassays, to identify patient's tumors that arelikely to respond to topo I targeted drugs. The topo I serine 506 assaycan be performed on cancer cells or tumor biopsies derived from thepatient, or other biological samples from the patient, such as blood,serum, urine, and/or sputum. The clinical application of the assay basedon phosphorylation of serine 506 can provide a straightforward andvaluable tool for identifying patients most likely to respond to suchtherapies, and for tailoring improved, individualized treatmentregimens. The assay can also be used in the neoadjuvant setting onbiopsy material to aid in the choice of therapy prior to surgery.

An antibody-based assay can be utilized in determining the sensitivityof a patient's cancer to a topoisomerase I inhibitor. The antibody-basedassay can provide direct identification of the phosphoserine 506epitope, an unambiguous indicator of a functional state of the topo Ithat is mechanistically linked to the generation of toxiccamptothecin-stablized cleavage complexes.

Alternative or complementary assays to detect CK2 protein, RNA, oractivity levels can provide additional indication of camptothecinsensitivity. For example, CK2 RNA expression can be evaluated bysemi-quantitative and quantitative PCR across a panel of samples withpreviously characterized topo I phosphorylation status and camptothecinsensitivity. Protein analysis of CK2 protein may be carried out byWestern analysis. RNA can be isolated from formalin-fixed,paraffin-embedded tissues as described in Korbler, et al., Experimentaland Molec Pathol 2003; 74:336-340, or from frozen tissue as described inHuang, et al., J Cell Mol Med 2009; 13:398-409. Semi-quantitative orquantitative PCR can be carried out as described in Huang, et al., JCell Mol Med 209; 13:398-409, using CK2 primers described in Kramerov,et al., Am J Pathol 2006; 168:1722-1736.

In one embodiment of the assay for determination of sensitivity of acancer cell to a topoisomerase I inhibitor, the status ofphosphorylation on serine 506 amino acid residue of topoisomerase I canbe visually identified. For example, to prepare an ELISA assay,polystyrene microtiter plates can be coated with 50 μl of varyingconcentrations (10 μg/ml to 50 μg/ml) of goat anti-topo-1 (in high pHbicarbonate buffer) as the capture antibody, following proceduresdescribed in Dudouet, et al., Cancer Res 1990; 50:438-443. Followingwashing, plates can be incubated with extracts of patient tumor cells(obtained from a biopsy) or patient blood, serum, urine, and/or sputum.This may be followed by treatment with either purified rabbit anti-topoI phosphoser 506 IgG or with anti-topo I nonphosphoser 506 IgG.Alternatively, mouse monoclonal IgGs to these epitopes can be used. Theinteraction can be detected colorimetrically using a biotinylatedanti-rabbit IgG (for example, Rabbit Link, Biogenix, San Ramon, Calif.)and streptavidin-conjugated Horse radish peroxidase (HRP) (for example,from Biogenix, San Ramon, Calif.), which binds to the detectionantibody. Finally, a colorimetric HRP substrate, o-phenylenediaminedihydrochloride can be added for 20 minutes, yielding a yellow productdetected by absorbance at 492 nm. The reaction may be stopped by theaddition sulfuric acid. As alternative approaches, one can employ theser506 epitope-specific rabbit antibodies as the capture antibodies,followed either by goat or mouse anti-topo I followed by the appropriatebiotinylated detection antibody.

An immunofluorescence-based or immunohistochemistry-based assayapplicable to frozen or paraffin-embedded tumor biopsies can also beused. This approach requires minimal amounts of material, and allows forthe detection of minor subpopulations that could be missed in pooledsamples, offering an advantage in certain settings. Frozen sections canbe processed using the methods previously detailed (see Lee, C., et al.,Cancer Res 2005; 65:9834-9842), involving fixation with formaldehyde,permeabilization with non-ionic detergent, and partial denaturation ofproteins with SDS to allow for exposure of internal epitopes (proceduresfor denaturation have been described in Donaldson, J. R., et al., 1998,Current Protocols in Cell Biology pp. 4.3.1 to 4.3.6, John Wiley andSons, Inc). Primary antibody treatment can then be Carried out, e.g.,with polyclonal rabbit anti topo I phospho ser506), or anti-topo Inonphospho ser 506, or with a general rabbit anti-topo I antibody,followed by secondary antibody staining with goat anti-rabbit IgG Alexafluor 486 (for example from Molecular Probes, Inc., Eugene, Oreg.).Controls can include secondary antibody only. Slides can becounterstained with the nuclear stain Hoescht 33342, mounted withcoverslips and examined by fluorescence microscopy.

For paraffin-imbedded sections, one can use immunohistochemicalprocedures described in Lebedeva, et al. Human Gene Therapy 2001;12:762-772. Briefly, slides can be deparafinized at 60° C. for 1 hour,followed by rehydration by sequential passage through xylene, ethanol(100%, 95%, 80%), H₂O, and PBS. They can then be treated with 3% H₂O₂,blocked with Superblock (Scytec Laboratories, Logan, Utah), and treatedwith the desired rabbit polycloncal primary IgG, followed by treatmentwith biotinylated goat anti IgG (for example, Multilink, Biogenix, SanRamon, Calif.) followed by treatment with streptavidin-conjugated horseradish peroxidase (HRP) (for example, from Biogeneix). Slides can thenbe incubated with the HRP substrate 3-amino-9-ethylcarbazole for coloreddevelopment. They can then be rinsed and mounted with coverslips with astandard aqueous mounting medium.

One or more samples of cancer cells can be screened for identificationof each sample as being sensitive or resistant to a topoisomerase Iinhibitor. When phosphorylation of serine 506 amino acid residue oftopoisomerase I in one sample is above a predetermined threshold asdetermined by the assay, that sample can be identified as beingsensitive to the topoisomerase I inhibitor. Conversely, whenphosphorylation of serine 506 amino acid residue of topoisomerase I inanother sample is below the predetermined threshold as determined by theassay, that sample can be identified as being resistant to thetopoisomerase I inhibitor. The predetermined threshold may be a ratio ofunphosphorylated topoisomerase I to phosphorylated topoisomerase Iwithin the cancer cells of a sample. In this way, a cancer cell samplecan be identified as being sensitive to the topoisomerase I inhibitorwhen the ratio is less than 1, and another cancer cell sample can beidentified as being resistant to the topoisomerase inhibitor I when theratio is greater than 1.

In an antibody based assay, the presence or absence of phosphorylationon serine 506 amino acid residue of topoisomerase I can determined by anantibody that binds phosphorylated serine 506 amino acid residue oftopoisomerase I, but does not bind nonphosphorylated serine 506 aminoacid residue of topoisomerase I. Alternatively, the presence or absenceof phosphorylation on serine 506 amino acid residue of topoisomerase Ican determined by an antibody that binds unphosphorylated serine 506amino acid residue of topoisomerase I, but does not bind phosphorylatedserine 506 amino acid residue of topoisomerase I. The antibody basedassay may utilize monoclonal or polyclonal antibodies disposed todetermine the presence or absence of phosphorylation on serine 506 aminoacid residue of topoisomerase I. By way of example, monoclonalantibodies can be produced to phosphorylated serine 506 epitope and alsoto non-phosphorylated serine 506 epitope, and their abilities can betested to identify cell lines with high and low topo I activity and/orhigh and low sensitivity to camptothecin, respectively, using immunoblot(Western), ELISA, and immunofluorescence of fixed cells (Wong andBerkenblit, Oncologist 2004; 9:68-79). Preferably, in order to provide aconsistent and stable supply of antibody, a mouse monoclonal to thephosphorylated serine 506 epitope can be generated following standardprocedures (Harlow, E., Lane, D. (1999) Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory; Nelson, P. N., Reynolds, G. M.,Waldron, E. E., Ward, E., Giannopoulos, K., and Murray, P. G. (2000)Monoclonal antibodies. Mol Pathol 53, 111-117).

Treatment of Cancer Patients

The determination of the status of phosphorylation on serine 506 aminoacid residue of topoisomerase I can be utilized in other applications aswell, such a treatment of cancer patients. The status of phosphorylationon serine 506 amino acid residue of topoisomerase I can be determined byassaying a biological specimen from the patient, e.g., tumor cells,tumor tissue, blood, serum, urine, and/or sputum. In this way, thepatient can be identified as being sensitive to a topoisomerase Iinhibitor when phosphorylation of serine 506 amino acid residue oftopoisomerase I is above a predetermined threshold as determined by theassay. Patients identified as being sensitive can then be administeredwith the topoisomerase I inhibitor as part of cancer treatment.

For cancer patients who are less sensitive or resistant to thetopoisomerase I inhibitor therapy, increasing their sensitivity to thetopoisomerase inhibitor can be included in their treatment. For example,a CK2 activator can be administered to the cancer patient in an amountsufficient to decrease ratio of unphosphorylated topoisomerase I tophosphorylated topoisomerase I, which may make the patient moresensitive to the topoisomerase I inhibitor. An exemplary dose may be inthe range of between about 2 mg to about 20 mg per kg of body weight,equivalent to between about 70 mg to about 700 mg per meter squared ofbody surface area. A preferred amount can be about 100 mg per metersquared of body surface area. For example,1-ethyl-4,5-dicarbamoylimidazole is one CK2 activator that may beutilized for the treatment of cancer patients that are less sensitive orresistant to the topoisomerase I inhibitor therapy.

EXAMPLES Example 1 Defective ARF/Topoisomerase I Complex Formation inH23 Cells

FIG. 1A shows a silver stained gel following a pull-down assay in whichimmobilized human ARF-thioredoxin fusion protein (or the N-terminaldomain (1-64) of ARF) was used to compare ARF-binding proteins fromDU145 (prostate cancer), H358, and H23 (non-small cell lung carcinoma)cell RIPA lysates.

Topoisomerase I bound to full-length ARF (ARF, FIG. 1A) but not the ARFN-terminal domain (ARF-N-term, amino acid residues 1-64, FIG. 1A)encoded by ARF's first exon (exon 1β). This is consistent with previousreports that topoisomerase I binds to ARF through the ARF C-terminal,exon 2-encoded domain (Ayrault, et al., Oncogene 2006; 25(19):2827(correction); Olivier, et al., Oncogene 2003; 22(13):1945-54). H23 cellsappeared to have significantly less topoisomerase I activity compared tothat measured in H358 cells (FIG. 1A, far right lane).

Western blot analysis confirmed that the level of topoisomerase I wasreduced in the fraction pulled down by immobilized ARF from H23 cellscompared to H358 cells (FIG. 1B, left panel). However, total endogenoustopoisomerase protein levels in H23 and H358 cells RIPA lysates weresimilar (FIG. 1B, right panel). Furthermore, a complete sequenceanalysis of the 2,295 base pair coding sequence of topoisomerase I inH23 cells showed that the sequence corresponded to the wild-typetopoisomerase I sequence (EC.5.99.1.2, Accession # NM_(—)003286). Thus,reduced binding of topoisomerase I from H23 cells to immobilized ARF isnot the result of reduced cellular levels of topoisomerase I nor is itthe result of a mutation in topoisomerase I that could alter its bindingproperties.

FIG. 1C shows the results of a co-immunoprecipitation experiment usingDNAse I solubilized nuclear extracts. This cellular fraction containsmore than 95% of topoisomerase I and ARF (Ayrault, et al., Oncogene2004; 23(49):8097-104). ARF-topoisomerase I complexes were readilydetectable in H358 nuclear extracts, but were undetectable in H23nuclear extracts (left panel, FIG. 1C). Thus, the failure oftopoisomerase I from H23 cell lysates to bind immobilized ARF isreflected in the lack of endogenous ARF/topoisomerase I complexes.

To determine whether overexpressed ectopic ARF could drive topoisomeraseI into complexes with ARF in H23 and H358 cells, cells were treated withan Adp14 adenoviral vector (moi, 20 pfu/cell) for 4 hours and preparednuclear extracts 48 hours later. Co-immunoprecipitation analysisfollowed by Western analysis showed that ARF-topoisomerase I complexesincrease about 3 fold in H358 cells following treatment with Adp14;indicating that not all cellular topoisomerase I had been bound by ARFin untreated cells (FIG. 1C middle panel). ARF-topoisomersase Icomplexes remained undetectable in H23 cells (FIG. 1C middle panel).

The material that remained unbound following two successiveimmunoprecipitations with anti-topoisomerase I was also analyzed (FIG.1C, right-hand panel (unbound)). Undetectable amounts of ARF protein inH358 cells were found in the unbound material, indicating that virtuallyall cellular ARF was complexed with topoisomerase I. In contrast, in H23cells, virtually all the cellular ARF was found in the unbound material.Taken together, these result demonstrate that the failure of H23 cellsto form ARF-topoisomerase I complexes, which are required fortopoisomerase I activity, are not a result of reduced ARF ortopoisomerase levels, nor are they a result of inactivating mutations ineither protein.

Vectors: The Adp14 vector encoding full-length ARF, the Ad1β vectorencoding the 64-amino acid residue N-terminal domain of ARF (ARFN-term), and vector treatment conditions have been described(Saadatmandi, et al., Cancer Gene Ther 2002; 9(10):830-9; Huang, et al.,Cancer Research 2003; 63:3646-3653). Equal titers of Adp14 and Ad1β wereconfirmed by RT-PCR to produce equivalent levels of ARF and ARF N-termmessage. An siRNA expression plasmid specific for the exon 2-encodedregion of ARF (pKD-Ink4a-v2), as well as a negative control siRNAexpression plasmid (pKD-NegCon-yl) were purchased from Upstate,Temecula, Calif., and transfected into cells using Lipofectamine™(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. An siRNA to the exon 113 region of ARF (sense sequence:5′-GGGUUUUCGUGGUUCACAUtt-3′ (SEQ ID NO: 4); antisense sequence:5′-AUGUGAACCACGAAAACCCtc-3′ (SEQ ID NO: 5)) was purchased from Ambion,Inc., Austin Tex.

Co-Immunoprecipitation/Western: DNAse I-solubilized nuclear extractswere prepared according to reference (Ayrault, et al., Oncogene 2004;23(49):8097-104). Briefly, cells (10⁶) were harvested and lysed in DNAseI solubilization buffer (10 mM Hepes pH 7.5, 100 mM NaCl, 300 mMsucrose, 3 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 1 mMphenylmethylsulfonyl fluoride (PMSF), complete protease inhibitors(Roche, Nutley, N.J.), followed by centrifugation to pellet nuclei.Nuclei were resuspended in 300 μl. of the same buffer, treated with 1mg/mL DNAse I (Sigma, St. Louis, Mo.) for 15 minutes at 37° C., andcentrifuged. The DNAse I-solubilized material, which contained the bulkof cellular topoisomerase I and ARF protein (Ayrault, et al., Oncogene,2004; 23(49):8097-104), was used for immunoprecipitation. (We found thathigh salt-extracted nuclei (see subnuclear fractionation andtopoisomerase I assays, below) and DNAse I-solubilized nuclei weresimilar with respect to topoisomerase I and ARF recovery; however, DNAseI solubilization avoided the use of high salt concentrations that woulddisrupt complexes). Co-immunoprecipitation was carried out in 1 mL ofthe same buffer, overnight at 4° C. with rocking, containing 175 μgprotein and 20 μL of antibody following our previously describedprocedure (Lee, et al., Cancer Res 2005; 65(21):9834-42). Where boundand unbound fractions were to be compared, the extracts were subjectedto 2 successive treatments with antibody (anti-topoisomerase I oranti-NPM), were found to be sufficient to deplete extracts ofimmunoreactive material. The immunoprecipitated material from the 1^(st)and 2^(nd) treatments was pooled and designated “bound”.Immunoprecipitates were incubated an additional hour in the presence of80 μL protein G agarose (Santa Cruz Biotechnology), centrifuged andwashed with PBS, resuspended in SDS-PAGE sample buffer, boiled,electrophoresed on a 12.5% SDS-PAGE gel, and subjected to Westernanalysis as described previously (Saadatmandi, et al., Cancer Gene Ther2002; 9(10):830-9). The material that did not immunoprecipitate wasdesignated “unbound” and was concentrated by precipitation with 5volumes of acetone, prior to resuspension in sample buffer. Antibodieswere: Goat polyclonal anti-topoisomerase I (Santa Cruz Biotechnology,Santa Cruz, Calif.), mouse monoclonal anti-nucleophosmin (NPM, B23)(Sigma, St. Louis, Mo.), rabbit polyclonal anti-full length ARF (ZymedLaboratories, Inc, South San Francisco, Calif.), mouse monoclonalanti-phosphoserine (Sigma, St. Louis, Mo.). All primary antibodies wereused at 1:100 for Westerns. Secondary antibodies for Westerns were goatanti-rabbit, goat anti-mouse, and donkey anti-goat (all purchased fromSanta Cruz Biotechnology, Santa Cruz, Calif.) and were used at 1:1000.

Subnuclear fractionation: Isolation of nuclei and preparation of nuclearextracts were carried out as described in reference (Olnes, et al.,Biotechniques 1994; 17(5):828-9), by swelling cells in hypotonic buffer(10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5mM Phenylmethylsulfonyl fluoride (PMSF), complete protease inhibitors(Roche, Nutley, N.J.)), and lysing cells by adding 0.6% NP40 to thehypotonic buffer, followed by centrifugation to recover nuclei. For thetopoisomerase I assays, nuclei were then extracted for 1 hour on ice inhigh salt buffer (20 mM Hepes pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA,1 mM DTT, 1 mM PMSF, 10% glycerol, and complete protease inhibitors).For storage, nuclear extracts were adjusted to 50% glycerol and placedat −80° C. until used. For preparation of nucleolar and nucleoplasmicfractions, the NP40-prepared nuclei were centrifuged through sucrose,sonicated, and fractionated by centrifugation again through sucrose asdescribed in reference (Andersen, et al., Curr Biol 2002; 12(1):1-11).Nucleoli were recovered in the pellet, and the unpelleted material(nucleoplasm) was concentrated by precipitation with 5 volumes ofacetone.

Example 2 H23 Nuclear Extracts have Reduced Topoisomerase I Activitywhich Cannot be Stimulated by ARF

H23 and H358 nuclear extracts were compared for topoisomerase I activityin vitro, and investigated whether the activities could be stimulated bythe addition of recombinant thioredoxin-ARF. As shown in FIG. 2A, H358topoisomerase I was found to be more effective at relaxing supercoiledplasmid DNA than was H23 topoisomerase, achieving 50% relaxation atabout 0.06 μg nuclear extract per reaction, some 10-fold lower than theamount of H23 extract needed to achieve the same level of relaxation(0.6 μg extract per reaction). A typical electrophoretic profile of thereaction products with increasing amounts of nuclear extract is shown inFIG. 2B in which 0.32, 0.65, or 1.3 μg of H358 cell extract (lanes 1-3)or H23 (lanes 4-6) were added in each reaction. “r” is the relaxed(non-supercoiled) plasmid and “s” is the supercoiled form.

Similar assays were carried out using the amount of each respectivenuclear extract that produced a 50% conversion of supercoiled to relaxedform (0.06 and 0.6 μg extract protein per reaction for H358 and H23,respectively), and added increasing amounts of purified thioredoxin-ARF(3, 9, 27 ng). As a control, in separate assays, increasing amounts ofthioredoxin-ARF-N-terminus, which does not bind to topoisomerase I, wasadded. Thioredoxin-ARF enhanced the activity of H358 topoisomerase in adose-dependent manner (FIG. 2C, lanes 1-3), but had no effect on H23topoisomerase (FIG. 2C, lanes 4-6), as expected based on the inabilityof ARF to bind to H23 topoisomerase. Neither H358 not H23 topoisomeraseactivities responded to the addition of thioredoxin ARF-N-terminus atsimilar doses (FIG. 2C lanes 7-12).

Topoisomerase I assays: Assays were carried out using the TopoisomeraseI Assay Kit (Topogen, Fla.), according to the instructions provided withthe kit and using the high salt nuclear extracts prepared as describedabove. Briefly, 0.125 μg supercoiled plasmid DNA was incubated with0-1.3 μg of nuclear extract for 30 minutes at 37° C. The reaction wasstopped by adding stop loading dye supplied in the kit andelectrophoresed on a 1% agarose/TAE (10 mM TRIS-acetate/1 mM EDTA) geluntil the dye front had reached the bottom of the gel. The gel was thenstained for 20 minutes in 0.5 μg/mL ethidium bromide, destained indeionized water for 30 minutes, and electrophoresed for an additionalhour to enhance band visibility. The gel was photographed and bandintensities were analyzed digitally using a Kodak digital camera andanalysis software. Some assays were carried out with alkalinephosphatase-treated extracts, prepared as described below, or in thepresence of ARF or ARF1β thioredoxin fusion proteins (3, 9, 27 ng),prepared as described above (see pull-down assays).

Example 3 Topoisomerase I is Activated by Both Phosphorylation and ARFBinding

A topoisomerase I immunoprecipitation analysis followed by Westerndetection of phosphoserine revealed that H358 cells expressed aserine-phosphorylated topoisomerase I (FIG. 3A, lane 1, top row). Asimilar analysis of phosphotyrosine revealed no evidence for tyrosinephosphorylation (data not shown). Similar results were found in PC-3cells (data not shown). In contrast, serine-phosphorylated topoisomeraseI was only weakly detectable in H23 cells (FIG. 3A, lane 2, top row).

Treatment of both H358 and H23 nuclear extracts with alkalinephosphatase (AP) eliminated serine phosphorylation (FIG. 3A, lanes 3, 4,top row) and abolished their topoisomerase I activity in vitro (FIG. 3B,lanes 4-6 and lanes 13-15). The dephosphorylated topoisomerase I fromH358 cells could no longer be activated by addition of increasingamounts of ARF fusion protein (FIG. 3B, lanes 7-9). Furthermore, whiletopoisomerase I co-immunoprecipitated with ARF from untreated H358nuclear extracts (FIG. 3A, lane 1, middle row), it failed toco-immunoprecipitate with ARF from H358 nuclear extracts treated withalkaline phosphatase (FIG. 3A, lane 3, middle row). Topoisomerase Ifailed to co-immunoprecipitate with ARF from either untreated oralkaline phosphatase-treated H23 cell nuclear extracts (FIG. 3A, lanes2,4, middle row).

When alkaline phosphatase-treated extracts from either H358 or H23 cellswere treated with casein kinase II (CKII), a serine kinase, we observedrestoration of serine phosphorylation (FIG. 3A, lanes 5,6, top row) andrestoration of ARF/topoisomerase I complex formation (FIG. 3A, lanes5,6, middle row). Recovery of topoisomerase I followingimmunoprecipitation was the same in all cases (FIG. 3A, lanes 1-6,bottom row).

Alkaline phosphatase treatment of purified recombinant humantopoisomerase I, abolished serine phosphorylation (FIG. 3C, lane 1, toprow), and abolished its ability to bind recombinant ARF fusion protein(FIG. 3C, lane 1, middle row). But, serine phosphorylation and ARFbinding could be restored by treatment with casein kinase II (FIG. 3C,lane 2, top and middle rows, respectively). Recovery of topoisomerase Ifollowing immunoprecipitation was the same in all cases (FIG. 3C, lanes1, 2 bottom row). Finally, a topoisomerase I IP/Western analysis wascarried out on lysates of an additional cell line, HT29, of colonadenocarcinoma origin. The results revealed a reduced level of serinephosphorylated topoisomerase I that correlated with failure to bind ARF,a result similar to what was seen with H23 cells. Taken together, theresults establish that differences in topoisomerase I serinephosphorylation account for the differences in ARF/topoisomerase Icomplex formation in observed in the cell lines examined.

Casein kinase II assays: 10⁶ cells were harvested, resuspended in 400 μl10 mM Tris pH 7.4, and subjected to 3 cycles of freeze/thaw. 50 μg ofcell extract was assayed for casein kinase II (CKII) activity using theCKII Assay kit from Upstate (Temecula, Calif.), following proceduressupplied with the kit.

Example 4 Variable CKII Levels Account for the Differences inTopoisomerase Activity Among Cell Lines

Additional assays were performed to determine whether the reduced levelsof topoisomerase I serine phosphorylation in H23 and HT29 cells could bedue to a reduced cellular levels of casein kinase II (CKII). As shown inFIG. 3D, H23 cell lysates display some 7% of the CKII activity of H358,and HT29 cells display some 41% of the activity of H358. The resultsindicate that low levels of CKII activity are likely to be responsiblefor the reduced levels of topoisomerase I serine phosphorylation andreduced ARF/topoisomerase I complex formation in H23 and HT 29 cells.

Example 5 Phosphorylated Topoisomerase I Retains ARF in the Nucleolus

Western analyses of ARF in subnuclear fractions, as well asimmunofluorescence staining of ARF in fixed H358 and H23 cells wasperformed to assess whether the interaction between ARF andtopoisomerase I affects subnuclear distribution. For Western analyses,nuclei were prepared as for the topoisomerase I assay, followed eitherby salt extraction to obtain total nuclear proteins, or by furthersubfractionation into nucleoplasmic and nucleolar fractions.

FIG. 4A shows the results of Western analyses carried out on totalnuclear and subnuclear fractions. Topoisomerase I and ARF levels werecomparable in H358 and 1-123 cells (FIG. 4A, left lanes). Cytoplasmiclevels of ARF and topoisomerase I were low to undetectable (not shown).Topoisomerase I was concentrated in the nucleolar fraction in both H358and H23 cells (FIG. 4A, top row). While ARF was also concentrated in thenucleolar fraction in H358 cells, it appeared to be evenly distributedbetween nucleolar and nucleoplasmic fractions in H23 cells (FIG. 4A,bottom row). This result was confirmed by immunofluorescence microscopyof fixed cells (FIG. 4B).

Nuclei were stained with the DNA stain, Hoechst 33342, which is excludedfrom nucleolar regions (top panels). Using an anti-ARF antibody, ARF wasdetected in a predominantly nucleolar staining pattern in H358 cells(FIG. 4B, bottom left). By contrast, in H23 cells, anti-ARF staining wasfound across the entire nuclear and perinuclear region (FIG. 4B, bottomright). Thus, failure of ARF to bind topoisomerase I correlates withdelocalization of ARF throughout the nucleus, suggesting thattopoisomerase contributes to the tethering of ARF in the nucleolus.

The interaction between ARF and nucleophosmin (NPM, B23), an abundantnucleolar protein, was examined in H358 and H23 cells. Nuclear extractsof H358 and H23 cells were immunoprecipitated with two successivetreatments with anti-NPM antibody, followed by Western detection of NPMand ARF in the pooled immunoprecipitated material (bnd) or in thematerial that remained unbound following two successiveimmunoprecipitations (un).

In H358 cells, virtually all of the cellular ARF was recovered in thematerial that co-immunoprecipitated with NPM, with undetectable levelsrecovered in the unbound material (FIG. 4C). This result is consistentwith a previous report in murine fibroblasts that the majority ofcellular ARF is bound to NPM (Bertwistle, et al., Mol Cell Biol 2004;24(3):985-96). In H23 cells, however, ARF was detected in approximatelyequivalent levels in the NPM-bound and unbound fractions (FIG. 4C),consistent with its decreased nucleolar localization. Because themajority of cellular ARF in H358 cells could also be recovered incomplexes with topoisomerase I (FIG. 1C, compare left and right panels),it is possible that topoisomerase I, ARF, and NPM are present togetherin a larger complex in H358 cells, and that defective binding of ARF totopoisomerase I in H23 cells destabilizes other interactions of ARFwithin the complex, including the interaction with NPM. Taken together,the results indicate that binding of ARF to topoisomerase I is requiredto maintain ARF's full nucleolar localization and its interaction withNPM.

Example 6 ARF Mediates Sensitivity to Topoisomerase I Inhibitors

Adenoviral vectors were used to achieve ectopic overexpression offull-length ARF (Adp14) or ARF-N-terminal domain (Ad1β), and RNAinterference to down-regulate endogenous expression of ARF. As shown bythe Western analysis of H358 cells in FIG. 5A, ARF levels increased bysome 3-fold, as determined by digital analysis of band intensities, by48 hours post-treatment with Adp14 (moi, 20 pfu/cell, FIG. 5A, lane 1),relative to Ad1β-treated cells (FIG. 5A, lane 2) or untreated cells(FIG. 5A, lane 4). By 72 hours post-transfection of an siRNA expressionplasmid to ARF exon 2 (FIG. 5A, lane 5), endogenous ARF levels fell to0.27 that found in untreated cells (FIG. 5A, lane 4) or controlsiRNA-treated cells (FIG. 5A, lane 3).

Viability assays were performed 24 hours post-vector treatment byexposing cells for 24 hours to increasing doses of camptothecin (atopoisomerase I inhibitor) in triplicate in a 96-well viability assay,and assaying them for viability 5 days post-start of vector treatment(FIG. 5B). For each growth curve, cell viabilities were normalized tothe viability of cells treated with vector only (no camptothecin), toenable a direct visualization of the sensitization effect. As shown forH358 cells in FIG. 5B (left assay), treatment of cells with Adp14resulted in a greater decrease in cell viability with increasingcamptothecin concentrations than did camptothecin alone, while treatmentof cells with siRNA to reduce ARF expression resulted in a smallerdecrease in cell viability with increasing camptothecin concentrations.Ad1β-treated cells overexpressing the ARF-N-terminal domain that doesnot interact with topoisomerase I, and control siRNA-treated cells inwhich levels of endogenous ARF remained unaltered, displayedcamptothecin responses similar to cells receiving no vector treatment(FIG. 5B, left).

To verify the generality of these observations, the same series ofassays were carried out with the PC-3 prostate cancer cell line (FIG.5B, right), with similar results. PC-3 cells express active, serinephosphorylated topoisomerase I (data not shown). The siRNA used todown-regulate endogenous ARF, targets the exon 2-encoded region of ARFthat is shared by the p16INK4A tumor suppressor. While H358 cellsexpress endogenous p161NK4A, PC-3 cells do not (Chi, et al., Clin CancerRes 1997; 3(10):1889-97), and they therefore provide a control showingthat the observed effect on camptothecin sensitivity can be attributedto ARF, and is not cell specific. As a further siRNA control, we reducedendogenous ARF expression in H358 cells by treating them with an siRNAto exon 1β, which is not shared with p161NK4A, and then restored ARFexpression by treatment with Adp14 one day later. As shown in theWestern analysis in FIG. 5C, siRNA treatment (lane 3, ARF) reduced ARFprotein levels to about 0.25 that of untreated cells (lane 1, ARF) by 72hours post-siRNA treatment. Digital analyses of ARF band intensities areshown below the ARF lanes. Treatment with Adp14 (moi, 100 pfu/cell) 24hours after siRNA treatment, restored ARF expression, measured 72 hourspost-siRNA treatment, to 1.3-fold that found in untreated cells (lane 2,ARF). Actin levels remained unchanged by these treatments (FIG. 5C,actin).

To assay how these treatments affected camptothecin responses,non-vector-treated cells, siRNA-treated cells, and siRNA+Adp14-treatedcells, were exposed to increasing doses of camptothecin as in FIG. 5Band assayed for viability 5 days post-start of vector treatment. Asshown by the viability assay in FIG. 5C, reduction in ARF expression inH358 cells following exon 1β siRNA treatment, resulted in decreasedsensitivity to camptothecin, while restoration and moderateoverexpression of ectopic ARF slightly enhanced sensitivity, supportingthe results in FIG. 5B.

Increased camptothecin sensitivity of Adp14-treated H358 cellscorrelated with about a 3-fold increase in ARF/topoisomerase I complexformation relative to Ad1β-treated, control siRNA treated, or non-vectortreated cells, as shown by the IP/Western analysis in FIG. 5D (upperpanel, lane 1 versus lanes 2-4), and with an increase in topoisomerase Iactivity (FIG. 5D, lower panel lane 1, bar 1 versus lanes 2-4, bars2-4). The decreased camptothecin sensitivity of siRNA-treated H358 cellscorrelated with about a 3-fold decrease in ARF/topoisomerase I complexformation (FIG. 5D, upper panel lane 5), and with a decrease intopoisomerase I activity (FIG. 5D, lower panel lane 5, bar 5).

The H23 cell line, with low to undetectable levels of endogenousARF/topoisomerase I complexes, respectively (see FIG. 1B) displayed agreatly reduced response to camptothecin (FIG. 5E), consistent withstudies showing that loss of topoisomerase I phosphorylation reducesactivity (Pommier, et al., J Biol Chem 1990; 265(16):9418-22). The factthat H23 cells cannot be sensitized to camptothecin by ectopicoverexpression of ARF indicates that ARF-mediated sensitization requiresits interaction with active, serine phosphorylated topoisomerase I.

Example 7 ARF Promotes Topoisomerase I DNA Binding

Topoisomerase I/DNA binding assay were performed to address themechanism by which ARF activates topoisomerase I. In FIG. 6 shows theresults from an immunodepletion assay in which topoisomerase I wastrapped in a complex with DNA by treatment of cells with camptothecin,followed by Western analysis of nuclei prepared with NP40. Becausetopoisomerase I/DNA complexes are too large to enter the gel, anincrease in topoisomerase I/DNA complex formation leads to a decrease inthe intensity of the topoisomerase I immunoreactive band representingnon-DNA-bound topoisomerase I. Treatment of H358 cells with increasingdoses of Adp14 resulted in a progressive decrease in non DNA-boundtopoisomerase I (FIG. 6 top panel), under conditions whereco-immunoprecipitated ARF/topoisomerase I complexes, released from NP40nuclei by DNase I treatment, increased (FIG. 4A, middle panel), andtotal topoisomerase I, released from NP40 nuclei by DNase I treatment,remained constant (FIG. 6, bottom panel). Thus increasedARF/topoisomerase I complex formation is accompanied by an increase inDNA bound topoisomerase I. The numbers below the lanes indicate digitalanalyses of band intensities, relative to the control lane (far left, 0moi Adp14).

Example 8 Camptothecin Sensitivity Correlates with Topoisomerase IPhosphorylation and with ARF Binding

FIG. 7 shows the results of an additional topoisomerase Iimmunoprecipitation (IP)/Western analyses as in FIG. 3, and cellviability assays in the presence of camptothecin. This experiment wasperformed to confirm the relationship between topoisomerase I serinephosphorylation, ARF/topoisomerase I complex formation, and cellularcamptothecin sensitivity. The Western blots were analyzed digitally andthe band intensities relative to H358 are plotted as bar graphs in FIG.7A-7C).

The PC-3 cell line displays a level of topoisomerase I serinephosphorylation similar to H358 (FIG. 7A), a similar level of totalcellular topoisomerase I (FIG. 7B), a similar level of cellular ARF/topI complex formation (FIG. 7C), and a similar degree of sensitivity tocamptothecin (FIG. 7D). In contrast, both H23 and HT29 cells display areduced level of topoisomerase I serine phosphorylation compared to H358(FIG. 7A), although total cellular topoisomerase I is similar to that ofH358 and PC-3 (FIG. 7B). H23 and HT 29 cells display reduced levels ofcellular ARF/topoisomerase I complex formation (FIG. 7C), and are moreresistant to camptothecin that are H358 and PC-3 cells (FIG. 7D).

Example 9 Hela Cells have Partially Defective ARF-Topoisomerase IComplex Formation and Show Intermediate Sensitivity to Camptothecin

Hela cells display a sensitivity to camptothecin intermediate to that ofH23 and H358 (FIG. 8A). The assay in FIG. 8A was carried out as in FIG.7D. ARF/topoisomerase I complex formation was examined using theco-immunoprecipitation assay described for FIGS. 3A and 3C. A reducedbut detectable level of ARF/topoisomerase I complex formation in Helacells was observed, compared to H358 cells (FIG. 8B). ARF/topoisomeraseI complex formation in H23 cells was undetectable (FIG. 8B), confirmingprevious experiments (FIG. 1C). However, topoisomerase I was serinephosphorylated in Hela cells (FIG. 8C), indicating that other factorsare likely to be responsible for the failure to form ARF/topoisomerase Icomplexes. Total ARF levels in H23, H358, and Hela cells were found tobe similar (FIG. 8D). The results indicate that defectiveARF/topoisomerase I complex formation can result from cellular changesother than defective phosphorylation of topoisomerase I, and correlateswith increased resistance to camptothecin.

Example 10 Treatment of Cancer in a Human

A human patient diagnosed with cancer may be treated according to themethods and principles of this disclosure. For example, a patientdiagnosed with prostate cancer, lung cancer, colon cancer, or ovarian isadministered once each day for five days, by intratumoral injection, 10⁵to 10¹⁰ viral particles of an adenoviral vector containing nucleic acidencoding functional CMI, operably linked to a promoter. Subsequently,the patient is administered Irinotecan at 100 mg/meter² weekly for 4weeks. This treatment regimen results in a reduction in the size of theprostate, lung, colon, or ovarian tumor, or the level ofprostate-specific antigen in the blood, or both.

During the course of this treatment regimen, the prostate, lung, colon,or ovarian cancer cells contain both an elevated serine kinasebiological activity (caused by treatment with the CKII-containingadenoviral vector) and a topoisomerase inhibitor (i.e., acamptothecin-derived drug such as Ironotecan or Topotecan).

Example 11 Camptothecin Sensitivity of Normal and Cancer-Derived CellLines and Correlation with Topo I Phosphorylation and CK2 but not PKC orcdk1 Activity and Protein Levels

Studies of a large panel of cell lines have shown that cell lines withoverexpressed CK2 (FIG. 11B1-B3 and 11C1-C3) display hyper serinephosphorylation of topo I (FIG. 11D) that correlates with sensitivity tocamptothecin (FIG. 11A). The cellular levels of two other serinekinases, PKC and cdk1, both of which have been implicated in topo Iserine phosphorylation, do not correlate with sensitivity tocamptothecin (FIG. 11A-C). Referring to FIG. 11A, 3-day viability assayscarried out in 96 well plates as described in Saadatmandi et al (2002)Cancer Gene Therapy 9:830-839. Cells were plated at 2000 cells per welland allowed to attach. Triplicate wells were then treated with theindicated doses of camptothecin for 18 hours, and monitored forviability 3 days post-start of treatment. Viability is represented as a% of control, untreated, cell viability. Referring to FIGS. 11B1-B3, PKC(B1), cdk1 (B2), and CK2 (B3) levels in cancer cell lines: Cell lysateswere prepared from the indicated cancer cell lines and assayed forenzymatic activities, or evaluated by Western analysis for total PKC,cdk1, or CK2 protein levels. Actin levels served as a control (samecontrol for PKC, cdk1, and CK2). Referring to FIGS. 11C1-C3 PKC (C1),cdk1 (C2), and CK2 (C3) levels in normal cell lines, compared to H358and H23: Cell lysates were prepared from the indicated cell lines andevaluated as before. Numbers below Western blots indicate digitalreading of band intensities of PCK, cdk1, or CK2 Western blots, relativeto H358. Referring to FIG. 11D, topo-I immunoprecipitation followed bytopo-I Western (top row) or phosphoserine Western (bottom row). Numbersbelow Western blot are digital readings of ser-P band intensitiesrelative to H358.

Example 12 CK2 is Necessary and Sufficient to Maintain Topo IPhosphorylation, Enzymatic Activity, and Phosphorylation-Dependent Topo1 Molecular Interactions In Vivo

To determine whether cellular levels of CK2 have functional significancewith regard to topo I properties, we examined how experimentalmodulation of CK2 activity in two representative cell lines, namelycamptothecin sensitive H358 cells and camptothecin resistant H23 cells,affects topo I phosphorylation, topo I complex formation with p14ARF,topo I activity, and camptothecin-induced DNA damage.

We down-regulated CK2 activity in H358 cells either by treating themwith the highly selective CK2 inhibitor TBB(4,5,6,7-tetrabromobenzotriazole), which has minimal effects on PKC orcdk1 (Sarno, et al., Febs Lett 2001; 496:44-48), or by downregulatingCK2 expression using an siRNA mixture with specificity for the α and α′isoforms of the CK2 catalytic subunit. Conversely, we upregulated CK2activity in H23 cells by treating them with a the CK2 activator,1-ethyl-4,5-dicarbamoylimidazole (Reikhardt, et al., Neurosci BehavPhysiol 2003; 22:799-804). The activator has a specific effect onpurified CK2 activity, stimulating purified CK2 holoenzyme activity orCK2α catalytic subunit activity in vitro some 6-fold when used at theconcentration used to treat cells (10 nM), while having no effect oneither purified PKC and cdk1 activity or on endogenous PKC or cdk1activity in treated H23 cells (FIGS. 15G and 15H, respectively). Asshown by the bar graph in FIG. 15A, TBB or CK2 siRNA treatment of H358cells reduces CK2 activity 72 hours later to some 25% of levels inuntreated cells. A control scrambled sequence siRNA has no effect. CK2activator treatment of H23 cells results in a 4-fold increase in CK2activity relative to untreated H23 cells. A similar treatment of H358cells results in only a 10% increase in CK2 activity (data not shown),suggesting that topo I in untreated H358 topo I is nearly maximallyphosphorylated at potential CK2-targeted sites.

We carried out a Western analysis of CK2 protein (α subunit) in lysatesof H358 and H23 cells 72 hours following the start of various treatmentsin FIG. 15A. As shown in the Western blot in FIG. 15B, TBB treatment ofH358 cells has no effect on CK2 protein levels, as expected, but CK2siRNA treatment of H358 cells reduces CK2 protein levels to about 47% ofcontrol levels (as determined by digital analyses of band intensities).CK2 activator treatment of H23 cells also enhances the accumulation ofCK2 protein (FIG. 15B, lane 5), but not CK2α transcription (FIG. 15I),suggesting that the activator may promote increased translation orstabilization of CK2 protein though unknown mechanisms, in addition toits direct activation effect on purified CK2.

We then examined levels of total topo I protein, topo I serinephosphorylation, and topo I complex formation with p14ARF in H358 or H23cells, treated as in FIG. 15A, by carrying out anti-topo Iimmunoprecipitations of nuclear extracts of treated cells, followed byWestern analyses of topo I, phosphoserine, and p14ARF in theimmunoprecipitated material (FIG. 15C). We found that total topo Iprotein levels are not affected by these treatments and remain similarin H358 and H23 cells (FIG. 15C, top panel), while the level of topo Iphosphorylation and complex formation with p14ARF correlates with levelsof CK2 activity (FIG. 15C, middle and lower panels, respectively). TBBor CK2-siRNA treatment of H358 cells reduces topo I serinephosphorylation to about 10% that of untreated H358 cells (as determinedby digital quantitation of band intensities), indicating that themajority of topo I hyperphosphorylation is under CK2 control in thesecells. Conversely, CK2 activator treatment of H23 cells increases topo Iphosphorylation by some 4.7-fold relative to untreated H23 cells, asdetermined by digital quantitation of band intensities (FIG. 15C, middlepanel), making it roughly equivalent to endogenous topo Iphosphorylation levels in untreated H358 cells. Finally, a Westernanalysis of p14ARF in the topo I-immunoprecipitated material (FIG. 15C,bottom panel), shows that p14ARF/topo I complex formation occurs only inthe presence of hyperphosphorylated topo I in both untreated H358 andCK2 activator-treated H23 cells. The results indicate that CK2-mediatedphosphorylation has functional significance in vivo, consistent with ourprevious results in vitro (Bandyopadhyay, et al., Biochemistry 2007;46:14325-14334).

We confirmed that the changes in topo I phosphorylation statuscorrespond to the predicted changes in topo I activity by assayingnuclear extracts of H358 and H23 cells (untreated, or 72 hours after thetreatments in FIG. 15A) for their ability to convert a supercoiledplasmid “s” to a relaxed form “r”. FIG. 15D shows an ethidium bromidestained agarose gel following electrophoresis of the reaction productsobtained from these assays. Under conditions where untreated H358 cellnuclear extract converts virtually all supercoiled plasmid form torelaxed form (0.75 μg lysate per reaction), essentially none isconverted using nuclear extracts from H358 cells treated with TBB or CK2siRNA, indicating that topo I activity is effectively inhibited in vivoby treatments that inhibit CK2 activity. Conversely, under conditionswhere nuclear extract from untreated H23 cells is essentially inactive(0.75 μg lysates protein per reaction), nuclear extract from CK2activator-treated H23 cells converts virtually all of the supercoiledform to relaxed form, indicating that CK2 activation is sufficient toactivate topo I activity to levels observed in H358 cells. Takentogether, these results indicate that CK2 is necessary and sufficient tomaintain topo I activity and function in these cancer cells.

The activation and suppression of topo I activity is predicted toproduce a corresponding increase and decrease in camptothecin-inducedDNA damage. Human topo I acts by introducing a single strand break inthe DNA double helix via an intermediate covalent complex between theenzyme and DNA termed a “cleavable complex,” in which an active tyrosylresidue at position 723 in the C-terminal domain of topo I becomeslinked to the 3-end of the DNA break, leaving a 5′-OH on the other sideof the break (see Champoux, Annu Rev Biochem 2001; 70:3690413), review).The passage of the non-cleaved strand unwinds the DNA by one linkagenumber and is followed by a resealing of the single strand break andrelease of the enzyme. Camptothecin and related drugs interact with thecleavage complex and stabilize it, so that DNA unwinding, resealing andenzyme release is prevented (Covey, et al., Cancer Res 1989;49:5016-5022; Kjeldsen, et al., J Mol Biol 1992; 228:1025-1030; Koster,et al., Nature 2007; 448:213-217; Porter, and Champoux, Nucleic AcidsRes 1989; 17:8521-8532; Svejstrup, et al., J Mol Biol 1991;222:669-678). The single strand break can become a lethal double strandbreak upon passage of the replication fork (Hsiang, et al., Cancer Res1989; 49:5077-5082). This mechanism, which converts topoisomerase I intoa cellular poison, has been proposed to account for the cytotoxicity ofcamptothecin (Tsao, et al., Cancer Res 1993; 53:5908-5914), and explainswhy low levels of topoisomerase I, by limiting the frequency of cleavagecomplex formation, favor cell survival in the presence of camptothecin.

To determine levels of covalent cleavage complex formation, cellstreated as in FIG. 15A, were DNA-labeled 72 hours later by overnightincubation in [³H]-thymidine, followed by exposure to camptothecin tostabilize cleavage complexes, and K⁺/SDS precipitation of covalent topoI-DNA cleavage complexes as described in Olnes and Kurl, Biotechniques1994; 17:828-829.

Under these conditions of precipitation, only DNA covalently linked totopo I will co-precipitate with it. As shown by the bar graph ofco-precipitated [³H]-thymidine-labeled DNA in FIG. 15E,camptothecin-stabilized cleavage complexes are some 5 to 7-fold morefrequent in cells expressing the highest levels of CK2 andphosphorylated topo I, indicating that more topo I molecules becomeassociated with cellular DNA under these conditions.

Finally, since camptothecin treatment ultimately leads to the productionof double strand DNA breaks (DSBs) in growing cells, we examined how thevarious treatments in FIG. 15A affect camptothecin-mediated induction ofthe phosphorylated form of the histone variant, H2A.X (denoted γ-H2A.X),which accumulates at sites of DSBs (Rogakou, et al., J Cell Biol 1999;146:905-916). Total H2A.X served as a control. Camptothecin exposure wasfor 1 hour, initiated 72 hours after treatment of cells as in FIG. 15A.As shown in FIG. 15F, γ-H2A.X and hence, DNA double strand breaks,accumulates in camptothecin-treated cells expressing high levels of CK2and phosphorylated topo I, confirming that the increased cellularsensitivity to camptothecin correlates with increased DNA damage.

Example 13 Camptothecin Sensitivity in Cancer Cells is FunctionallyLinked to CK2 Activity

Referring to FIGS. 13A and 13B, graphs are shown for experimentsestablishing a functional relationship between CK2 and the cellularresponse to camptothecin, further validating CK2 as a biomarker fortherapy responsiveness. Experimental inhibition of CK2 incamptothecin-sensitive H358 cells makes these cells more resistant tocamptothecin (FIG. 13A), and conversely, experimental activation of CK2in camptothecin-resistant H23 cells makes them more sensitive tocamptothecin (FIG. 13B). Experimental inhibition of CK2 in H358 cellswas accomplished by pretreating them with 10 μM TBB(4,5,6,7-tetrabromobenzotriazole) for 1 hour (TBB purchased fromCalbiochem), or by transducing them with an siRNA against CK2 (purchasedfrom Upstate/Millipore). Both pretreatments were sufficient to reduceCK2 activity by about 75% over the 3-day period of the growth assaycompared to cells that received no pretreatment or cells that weretransduced with a control, scrambled siRNA. Experimental activation ofCK2 in H23 cells was accomplished by maintaining cells in the presenceof 10 nM of the CK2 activator, 1-ethyl-4,5-dicarbamoylimidazole(described in Reikhardt, et al. Neuroscience and Behavioral Physiology2003; 33:799-804). This was the lowest dose (over the range of 5-100 nM)that could activate CK2 while having no effect on cell viability.

Example 14 Novel Topoisomerase I Phospho Epitope IdentifiesCamptothecin-Sensitive Cancer Cell Lines

A novel CK2-mediated topo I phosphorylation site on serine 506 has beenidentified by a mass spectrometry analysis of purifiedbaculovirus-expressed recombinant human topo I followingdephosphorylation with alkaline phosphatase and extensiverephosphorylation with CK2. A rabbit polyclonal antibody was generatedto a topo I peptide containing phosphoserine 506 (the sequence of theimmunizing phosphopeptide is as follows:H-Thr-Val-Gly-Cys(Acm)-Cys(acm)-pSer-Leu-Arg-Val-Glu-His-Ile-Asn-Leu-His-Pro-Glu-Leu-lys-Lys-Cys-NH2).A control antibody was generated to the unphosphorylated peptide. Thepurified anti-topo I phosphoserine 506 IgG was found to beimmunoreactive on Western blots with recombinant, baculovirus-expressedtopo I that had been dephosphorylated with alkaline phosphates andrephosphorylated with CK2, but not with dephosphorylated topo I (FIG.14A).

The anti-topo I phosphoserine 506 IgG was also found to beimmunoreactive on Western blots with cellular topo I from thecamptothecin-sensitive non small cell lung cancer Cell line, H358, butnot with cellular topo I from the camptothecin-resistant non small celllung cancer cell line, H23 (FIG. 14B). In contrast, the control IgGgenerated to the non-phosphorylated epitope reacted poorly with H383topo I but strongly with H23 topo I (FIG. 14B).

Analysis of a broader array of human cancer cell lines, and twoimmortalized cell lines derived from normal human epithelial cells(Het1A) or human fibroblasts (BJ-1), showed that the phospho-specificantibody recognized cellular topo I from those cell lines that displayedsensitivity to camptothecin (FIG. 11A), namely H358, PC-3, DU145, LnCAP,OC3, and MDA MB 435 (abbreviated MB435). The camptothecin resistantcancer cell lines, H23 and HT29, and immortalized normal cell lines,BJ-1 and HET1A, expressed equivalent levels of topo I protein but it didnot react with the phospho-specific antibody (FIG. 14C).

The phosphorylated epitope appears therefore to be a cancer-specifictopo I abnormality directly related cellular sensitivity to topoI-targeted drugs. Furthermore, the anti-topo I phosphoserine 506 IgG canbe used for immunofluorescence detection of phospho topo I in fixed,permeabilized H358 cells (FIG. 14D). This epitope can therefore beamenable to immunofluoresecence assays of fixed tumor specimens. Thisepitope is not present in normal cells, as shown by the Western blot inFIG. 14E of lysates of normal BJ1 human fibroblasts and normal HET1Ahuman epithelial cells. A functional relationship between CK2 and thephosphoserine 506 epitope was demonstrated in the Western blots in FIG.14F by showing that activation of CK2 in H23 cells treated with the CK2activator as in FIG. 13B induces cellular levels of the phosphorylatedepitope, detected with the anti-topo I phosphoserine 506 IgG (FIG. 14F,upper panel) and inhibition of CK2 in H358 cells treated with TBB as inFIG. 13A suppresses cellular levels of the phosphoserine epitope,detected with the anti-topo I phosphoserine 506 IgG (FIG. 14F, upperpanel). Total topoisomerase I levels served as a control (lower panel,FIG. 14F).

Example 15 CK2 mRNA Levels are Upregulated in Camptothecin-SensitiveCancer Cells

FIG. 12 shows the result of a semi quantitative PCR of CK2 mRNA levels.Analysis of CK2 mRNA levels in cellular RNA, normalized to levels inHET1A cells, showed that levels in normal cells (HET1A, BJ-1, GT41F) andthe 3 camptothecin-resistant cancer cell lines (H23, HT29, SW480) arelower than levels in the 6 camptothecin-sensitive cancer cell lines(H358, PC3, DU145, LnCAP, MDAMB-435, OC3). Digital quantitation of bandintensities for CK2 are shown below the lanes. RNA was isolated using aRNA isolation kit (Qiagen, Valencia, Calif.) and RT-PCR was performedusing CK2α primers and conditions described in Kramerov et al., Am JPathol, 2006; 168:1722-1736, which produce a 151 bp DNA fragmentrevealed by ethidium bromide-stained agarose gel electrophoresis. Actinamplification of parallel aliquots served as a control. The results showthat the CK2 activator did not act at the level of CK2α transcription.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation; andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understobd that although the invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

1. A method for determining the sensitivity of a cancer cell to atopoisomerase I inhibitor, comprising: (i) determining status ofphosphorylation on serine 506 amino acid residue of topoisomerase Iwithin said cancer cell by way of an assay; and (ii) identifying saidcancer cell as being sensitive to said topoisomerase I inhibitor whenphosphorylation of serine 506 amino acid residue of topoisomerase I isabove a predetermined threshold as determined by said assay, andidentifying said cancer cell as being resistant to the topoisomerase Iinhibitor when phosphorylation of serine 506 amino acid residue oftopoisomerase I is below the predetermined threshold as determined bythe assay.
 2. The method of claim 1, wherein the predetermined thresholdis a ratio of unphosphorylated topoisomerase I to phosphorylatedtopoisomerase I within said cancer cell.
 3. The method of claim 2,wherein said cancer cell is identified as being sensitive to thetopoisomerase I inhibitor when the ratio is less than 1, and said cancercell is identified as being resistant to the topoisomerase inhibitor Iwhen the ratio is greater than
 1. 4. The method of claim 1, wherein saidtopoisomerase I inhibitor is selected from the group consisting ofcamptothecin, irinotecan, topotecan, an analogue thereof, or anon-camptothecin-derived topoisomerase I inhibitor.
 5. The method ofclaim 1, wherein the presence or absence of phosphorylation on serine506 amino acid residue of topoisomerase I is determined by an antibodybased assay.
 6. The method of claim 5, wherein said antibody based assaycomprises an antibody that binds phosphorylated serine 506 amino acidresidue of topoisomerase I, but does not bind nonphosphorylated serine506 amino acid residue of topoisomerase I.
 7. The method of claim 5,wherein said antibody based assay comprises an antibody that bindsunphosphorylated serine 506 amino acid residue of topoisomerase I, butdoes not bind phosphorylated serine 506 amino acid residue oftopoisomerase I.
 8. The method of claim 7, wherein said antibodycomprises a polyclonal antibody.
 9. The method of claim 7, wherein saidantibody comprises a monoclonal antibody.
 10. The method of claim 1,further comprising evaluating CK2 RNA expression of the cancer cell as aconfirmatory diagnostic test.
 11. The method of claim 1, wherein saidcancer cell is selected from the groups consisting of a lung cancercell, a prostate cancer cell, a hepatocellular carcinoma cell, a breastcancer cell, a colorectal cancer cell, an acute myelogenous leukemiacell, a melanoma cell, an ovarian cancer cell, a neuroendocrinecarcinoma cell, a gastric cancer cell, an esophageal cancer cell, apancreatic cancer cell, an adenocarcinoma cell, a brain cancer cell, ahead and neck cancer cell, a bone marrow-derived cancer cell, a bonecancer cell, a kidney cancer cell, a retina cancer cell, a bladdercancer cell, a liver cancer cell, and a mesothelioma cancer cell.
 12. Amethod for treating cancer in a patient, comprising: (i) determiningstatus of phosphorylation, by way of an assay, on serine 506 amino acidresidue of topoisomerase I in a biological specimen from said patient;(ii) identifying said patient as being sensitive to a topoisomerase Iinhibitor when phosphorylation of serine 506 amino acid residue oftopoisomerase I is above a predetermined threshold as determined by saidassay; and (iii) administering the topoisomerase I inhibitor to saidpatient.
 13. The method of claim 12, wherein the predetermined thresholdis a ratio of unphosphorylated topoisomerase I to phosphorylatedtopoisomerase I within said biological sample.
 14. The method of claim13, wherein said patient is identified as being sensitive to thetopoisomerase I inhibitor when the ratio is less than 1, and saidpatient is identified as being resistant to the topoisomerase inhibitorI when the ratio is greater than
 1. 15. The method of claim 12, whereinthe biological specimen is selected from the group consisting of tumorcells, tumor tissue, blood, serum, urine, and sputum.
 16. The method ofclaim 12, wherein said topoisomerase I inhibitor is selected from thegroup consisting of camptothecin, irinotecan, topotecan, an analoguethereof, or a non-camptothecin-derived topoisomerase I inhibitor. 17.The methods of claim 12, wherein said cancer is selected from the groupsconsisting of lung cancer, prostate cancer, hepatocellular carcinoma,breast cancer, colorectal cancer, acute myelogenous leukemia, melanoma,ovarian cancer, neuroendocrine carcinoma, gastric cancer, esophagealcancer, pancreatic cancer, adenocarcinoma, brain cancer, head and neckcancer, bone marrow-derived cancer, bone cancer, kidney cancer, retinacancer, bladder cancer, liver cancer, and mesothelioma cancer.
 18. Amethod of increasing the sensitivity of a cancer patient to atopoisomerase inhibitor, comprising administering a CK2 activator tosaid cancer patient in an amount sufficient to decrease ratio ofunphosphorylated topoisomerase I to phosphorylated topoisomerase I. 19.The method of claim 18, wherein the ratio of unphosphorylatedtopoisomerase Ito phosphorylated topoisomerase I is decreased to lessthan
 1. 20. The method of claim 18, wherein said CK2 activator is1-ethyl-4,5-dicarbamoylimidazole.